Regeneration and repair of neural tissue using postpartum-derived cells

ABSTRACT

Cells derived from postpartum umbilicus and placenta are disclosed. Pharmaceutical compositions, devices and methods for the regeneration or repair of neural tissue using the postpartum-derived cells are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35 U.S.C 119(e) of U.S.Provisional Application No. 60/483,264, filed Jun. 27, 2003, the entirecontents of which are incorporated by reference herein.

This application is also related to the following commonly-owned,co-pending applications, the entire contents of each of which areincorporated by reference herein: U.S. application Ser. No. 10/877,012,filed Jun. 25, 2004, U.S. application Ser. No. 10/877,446, filed Jun.25, 2004, U.S. application Ser. No. 10/877,445, filed Jun. 25, 2004,U.S. application Ser. No. 10/877,541, filed Jun. 25, 2004, U.S.application Ser. No. 10/877,009, filed Jun. 25, 2004, U.S. applicationSer. No. 10/876,998, filed Jun. 25, 2004 and U.S. ProvisionalApplication No. 60/555,908, filed Mar. 24, 2004.

FIELD OF THE INVENTION

This invention relates to the field of cell-based or regenerativetherapy for neurological diseases and disorders. In particular, theinvention provides pharmaceutical compositions, devices and methods forthe regeneration or repair of neural tissue using postpartum derivedcells.

BACKGROUND OF THE INVENTION

Various patents and other publications are referred to throughout thespecification. Each of these publications is incorporated by referenceherein, in its entirety.

Neurological diseases and other disorders of the central and peripheralnervous system are among the most debilitating that can be suffered byan individual, not only because of their physical effects, but alsobecause of their permanence. In the past, a patient suffering from brainor spinal cord injury, or a neurodegenerative condition of the centralor peripheral nervous system, such as Parkinson's disease, Alzheimer'sdisease or multiple sclerosis, to name a few, held little hope forrecovery or cure.

Neurological damage and neurodegenerative diseases were long thought tobe irreversible because of the inability of neurons and other cells ofthe nervous system to grow in the adult body. However, the recent adventof stem cell-based therapy for tissue repair and regeneration providespromising treatments for a number of neurodegenerative pathologies andother neurological disorders. Stem cells are capable of self-renewal anddifferentiation to generate a variety of mature neural cell lineages.Transplantation of such cells can be utilized as a clinical tool forreconstituting a target tissue, thereby restoring physiologic andanatomic functionality. The application of stem cell technology iswide-ranging, including tissue engineering, gene therapy delivery, andcell therapeutics, i.e., delivery of biotherapeutic agents to a targetlocation via exogenously supplied living cells or cellular componentsthat produce or contain those agents (For a review, see Tresco, P. A. etal., 2000, Advanced Drug Delivery Reviews 42: 2-37).

An obstacle to realization of the therapeutic potential of stem celltechnology has been the difficulty of obtaining sufficient numbers ofstem cells. One source of stem cells is embryonic or fetal tissue.Embryonic stem and progenitor cells have been isolated from a number ofmammalian species, including humans, and several such cell types havebeen shown capable of self-renewal and expansion, as welldifferentiation into all neurological cell lineages. But the derivationof stem cells from embryonic or fetal sources has raised many ethicaland moral issues that are desirable to avoid by identifying othersources of multipotent or pluripotent cells.

Stem cells with neural potency also have been isolated from adulttissues. Neural stem cells exist in the developing brain and in theadult nervous system. These cells can undergo expansion and candifferentiate into neurons, astrocytes and oligodendrocytes. However,adult neural stem cells are rare, as well as being obtainable only byinvasive procedures, and may have a more limited ability to expand inculture than do embryonic stem cells.

Other adult tissue may also yield progenitor cells useful for cell-basedneural therapy. For instance, it has been reported recently that adultstem cells derived from bone marrow and skin can be expanded in cultureand give rise to multiple lineages, including some neural lineages.

Postpartum tissues, such as the umbilical cord and placenta, havegenerated interest as an alternative source of stem cells. For example,methods for recovery of stem cells by perfusion of the placenta orcollection from umbilical cord blood or tissue have been described. Alimitation of stem cell procurement from these methods has been aninadequate volume of cord blood or quantity of cells obtained, as wellas heterogeneity in, or lack of characterization of, the populations ofcells obtained from those sources.

Thus, alternative sources of adequate supplies of cells having theability to differentiate into an array of neural cell lineages remain ingreat demand. A reliable, well-characterized and plentiful supply ofsubstantially homogeneous populations of such cells would be anadvantage in a variety of diagnostic and therapeutic applications inneural repair and regeneration, including drug screening assays, ex vivoor in vitro trophic support of other neural cells, and in vivocell-based therapy.

SUMMARY OF THE INVENTION

This invention provides compositions and methods applicable tocell-based or regenerative therapy for neurological diseases anddisorders. In particular, the invention features pharmaceuticalcompositions, devices and methods for the regeneration or repair ofneural tissue using postpartum-derived cells.

One aspect of the invention features an isolated postpartum-derivedcell, derived from human placental or umbilical cord tissuesubstantially free of blood, wherein the cell is capable of self-renewaland expansion in culture and has the potential to differentiate into acell of a neural phenotype; wherein the cell requires L-valine forgrowth and is capable of growth in at least about 5% oxygen. This cellfurther comprises one or more of the following characteristics: (a)potential for at least about 40 doublings in culture; (b) attachment andexpansion on a coated or uncoated tissue culture vessel, wherein thecoated tissue culture vessel comprises a coating of gelatin, laminin,collagen, polyornithine, vitronectin, or fibronectin; (c) production ofat least one of tissue factor, vimentin, and alpha-smooth muscle actin;(d) production of at least one of CD10, CD13, CD44, CD73, CD90,PDGFr-alpha, PD-L2 and HLA-A,B,C; (e) lack of production of at least oneof CD31, CD34, CD45, CD80, CD86, CD117, CD141, CD178, B7-H2, HLA-G, andHLA-DR, DP, DQ, as detected by flow cytometry; (f) expression of a gene,which relative to a human cell that is a fibroblast, a mesenchymal stemcell, or an iliac crest bone marrow cell, is increased for at least oneof a gene encoding: interleukin 8; reticulon 1; chemokine (C-X-C motif)ligand 1 (melonoma growth stimulating activity, alpha); chemokine (C-X-Cmotif) ligand 6 (granulocyte chemotactic protein 2); chemokine (C-X-Cmotif) ligand 3; tumor necrosis factor, alpha-induced protein 3; C-typelectin superfamily member 2; Wilms tumor 1; aldehyde dehydrogenase 1family member A2; renin; oxidized low density lipoprotein receptor 1;Homo sapiens clone IMAGE:4179671; protein kinase C zeta; hypotheticalprotein DKFZp564F013; downregulated in ovarian cancer 1; and Homosapiens gene from clone DKFZp547k1113; (g) expression of a gene, whichrelative to a human cell that is a fibroblast, a mesenchymal stem cell,or an iliac crest bone marrow cell, is reduced for at least one of agene encoding: short stature homeobox 2; heat shock 27 kDa protein 2;chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1);elastin (supravalvular aortic stenosis, Williams-Beuren syndrome); Homosapiens mRNA; cDNA DKFZp586M2022 (from clone DKFZp586M2022); mesenchymehomeo box 2 (growth arrest-specific homeo box); sine oculis homeoboxhomolog 1 (Drosophila); crystallin, alpha B; disheveled associatedactivator of morphogenesis 2; DKFZP586B2420 protein; similar to neuralin1; tetranectin (plasminogen binding protein); src homology three (SH3)and cysteine rich domain; cholesterol 25-hydroxylase; runt-relatedtranscription factor 3; interleukin 11 receptor, alpha; procollagenC-endopeptidase enhancer; frizzled homolog 7 (Drosophila); hypotheticalgene BC008967; collagen, type VIII, alpha 1; tenascin C (hexabrachion);iroquois homeobox protein 5; hephaestin; integrin, beta 8; synapticvesicle glycoprotein 2; neuroblastoma, suppression of tumorigenicity 1;insulin-like growth factor binding protein 2, 36 kDa; Homo sapiens cDNAFLJ12280 fis, clone MAMMA1001744; cytokine receptor-like factor 1;potassium intermediate/small conductance calcium-activated channel,subfamily N, member 4; integrin, beta 7; transcriptional co-activatorwith PDZ-binding motif (TAZ); sine oculis homeobox homolog 2(Drosophila); KIAA1034 protein; vesicle-associated membrane protein 5(myobrevin); EGF-containing fibulin-like extracellular matrix protein 1;early growth response 3; distal-less homeo box 5; hypothetical proteinFLJ20373; aldo-keto reductase family 1, member C3 (3-alphahydroxysteroid dehydrogenase, type II); biglycan; transcriptionalco-activator with PDZ-binding motif (TAZ); fibronectin 1; proenkephalin;integrin, beta-like 1 (with EGF-like repeat domains); Homo sapiens mRNAfull length insert cDNA clone EUROIMAGE 1968422; EphA3; KIAA0367protein; natriuretic peptide receptor C/guanylate cyclase C(atrionatriuretic peptide receptor C); hypothetical protein FLJ14054;Homo sapiens mRNA; cDNA DKFZp564B222 (from clone DKFZp564B222);BCL2/adenovirus E1B 19 kDa interacting protein 3-like; AE bindingprotein 1; cytochrome c oxidase subunit VIIa polypeptide 1 (muscle);similar to neuralin 1; B cell translocation gene 1; hypothetical proteinFLJ23191; and DKFZp586L151; (h) secretion of at least one of MCP-1,IL-6, IL-8, GCP-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO, MIP1a, RANTES, andTIMP1; and (i) lack of secretion of at least one of TGF-beta2, ANG2,PDGFbb, MIP1b, I309, MDC, and VEGF, as detected by ELISA.

In certain embodiments, the postpartum-derived cell is anumbilicus-derived cell. In other embodiments it is a placenta-derivedcell. In specific embodiments, the cell has all identifying features ofany one of: cell type PLA 071003 (P8) (ATCC Accession No. PTA-6074);cell type PLA 071003 (P11) (ATCC Accession No. PTA-6075); cell type PLA071003 (P16) (ATCC Accession No. PTA-6079); cell type UMB 022803 (P7)(ATCC Accession No. PTA-6067); or cell type UMB 022803 (P17) (ATCCAccession No. PTA-6068).

In certain embodiments, postpartum-derived cells are isolated in thepresence of one or more enzyme activities comprising metalloproteaseactivity, mucolytic activity and neutral protease activity. Preferably,the cells have a normal karyotype, which is maintained as the cells arepassaged in culture. In preferred embodiments, the postpartum-derivedcells comprise each of CD10, CD13, CD44, CD73, CD90, PDGFr-alpha, andHLA-A,B,C and does not comprise any of CD31, CD34, CD45, CD117, CD141,or HLA-DR,DP,DQ, as detected by flow cytometry.

Another aspect of the invention features a cell population comprisingthe postpartum-derived cells as described above. In one embodiment, thepopulation is a substantially homogeneous population of thepostpartum-derived cells. In a specific embodiment, the populationcomprises a clonal cell line of the postpartum-derived cells. In anotherembodiment, the population is a heterogeneous population comprising thepostpartum-derived cells and at least one other cell type. In certainembodiments, the other cell type is an astrocyte, oligodendrocyte,neuron, neural progenitor, neural stem cell or other multipotent orpluripotent stem cell. In other embodiments, the cell population iscultured in contact with one or more factors that stimulate stem celldifferentiation toward a neural lineage.

Also featured in accordance with the present invention is a cell lysateprepared from postpartum-derived cells. The cell lysate may be separatedinto a membrane enriched fraction and a soluble cell fraction. Theinvention also features an extracellular matrix produced by thepostpartum-derived cells, as well as a conditioned medium in which thecells have been grown.

Another aspect of the invention features a method of treating a patienthaving a neurodegenerative condition, the method comprisingadministering to the patient postpartum-derived cells as describedabove, in an amount effective to treat the neurodegenerative condition.In certain embodiments, the neurodegenerative condition is an acuteneurodegenerative condition, such as a brain trauma, spinal cord traumaor peripheral nerve trauma. In other embodiments, it is a chronic orprogressive neurodegenerative condition, such as Parkinson's disease,Alzheimer's disease, Huntington's disease, amyotrophic lateralsclerosis, tumor, multiple sclerosis or chronic peripheral nerve injury.

In one embodiment, the postpartum-derived cells are induced in vitro todifferentiate into a neural lineage cells prior to administration. Inanother embodiment, the cells are genetically engineered to produce agene product that promotes treatment of the neurodegenerative condition.

In certain embodiments, the cells are administered with at least oneother cell type, such as an astrocyte, oligodendrocyte, neuron, neuralprogenitor, neural stem cell or other multipotent or pluripotent stemcell. In these embodiments, the other cell type can be administeredsimultaneously with, or before, or after, the postpartum-derived cells.Likewise, in these or other embodiments, the cells are administered withat least one other agent, such as a drug for neural therapy, or anotherbeneficial adjunctive agent such as an anti-inflammatory agent,anti-apoptotic agents, antioxidant or growth factor. In theseembodiments, the other agent can be administered simultaneously with, orbefore, or after, the postpartum-derived cells.

In certain embodiments, the cells are administered at a pre-determinedsite in the central or peripheral nervous system of the patient. Theycan be administered by injection or infusion, or encapsulated within animplantable device, or by implantation of a matrix or scaffoldcontaining the cells.

Another aspect of the invention features a pharmaceutical compositionfor treating a patient having a neurodegenerative condition, comprisinga pharmaceutically acceptable carrier and the postpartum-derived cellsdescribed above. The neurodegenerative condition to be treated may be anacute neurodegenerative condition, or it may be a chronic or progressivecondition.

In certain embodiments, the pharmaceutical composition comprises cellsthat have been induced in vitro to differentiate into a neural lineagecells prior to formulation of the composition, or cells that have beengenetically engineered to produce a gene product that promotes treatmentof the neurodegenerative condition.

In certain embodiments, the pharmaceutical composition comprises atleast one other cell type, such as astrocyte, oligodendrocyte, neuron,neural progenitor, neural stem cell or other multipotent or pluripotentstem cell. In these or other embodiments, the pharmaceutical compositioncomprises at least one other agent. such as a drug for neural therapy,or another beneficial adjunctive agent such as an anti-inflammatoryagent, anti-apoptotic agents, antioxidant or growth factor.

In certain embodiments, the pharmaceutical composition is formulated foradministration by injection or infusion. Alternatively, it may comprisean implantable device in which the cells are encapsulated, or a matrixor scaffold containing the cells.

According to yet another aspect of the invention, a kit is provided fortreating a patient having a neurodegenerative condition. The kitcomprises a pharmaceutically acceptable carrier, a population of theabove-described postpartum-derived cells and instructions for using thekit in a method of treating the patient. The kit may further comprisesat least one reagent and instructions for culturing thepostpartum-derived cells. It may also comprise a population of at leastone other cell type, or at least one other agent for treating aneurodegenerative condition.

According to another aspect of the invention, a method is provided fortreating a patient having a neurodegenerative condition, which comprisesadministering to the patient and a preparation made from theabove-described postpartum-derived cells. Such a preparation maycomprises a cell lysate (or fraction thereof) of the postpartum-derivedcells, an extracellular matrix of the postpartum-derived cells, or aconditioned medium in which the postpartum-derived cells were grown. Inanother aspect, the invention features a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and a preparation madefrom the postpartum-derived cells, which may be a cell lysate (orfraction thereof) of the postpartum-derived cells, an extracellularmatrix of the postpartum-derived cells or a conditioned medium in whichthe postpartum-derived cells were grown. Kits for practicing this aspectof the invention are also provided. These may include the one or more ofa pharmaceutically acceptable carrier or other agent or reagent, one ormore of a cell lysate or fraction thereof, an extracellular matrix or aconditioned medium from the postpartum-derived cells, and instructionsfor use of the kit components.

Another aspect of the invention features a method for increasing thesurvival, growth or activity of neural lineage cells. The methodcomprises co-culturing the neural lineage cells with thepostpartum-derived cells of the invention, under conditions effective toincrease the survival, growth or activity of the neural lineage cells. Akit for practicing the method is also provided. The kit comprises thepostpartum-derived cells and instructions for co-culturing the neurallineage cells with the postpartum-derived cells under conditionseffective to increase the survival, growth or activity of the neurallineage cells.

Other features and advantages of the invention will be apparent from thedetailed description and examples that follow.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Definitions

Various terms used throughout the specification and claims are definedas set forth below.

Stem cells are undifferentiated cells defined by the ability of a singlecell both to self-renew, and to differentiate to produce progeny cells,including self-renewing progenitors, non-renewing progenitors, andterminally differentiated cells. Stem cells are also characterized bytheir ability to differentiate in vitro into functional cells of variouscell lineages from multiple germ layers (endoderm, mesoderm andectoderm), as well as to give rise to tissues of multiple germ layersfollowing transplantation, and to contribute substantially to most, ifnot all, tissues following injection into blastocysts.

Stem cells are classified according to their developmental potential as:(1) totipotent; (2) pluripotent; (3) multipotent; (4) oligopotent; and(5) unipotent. Totipotent cells are able to give rise to all embryonicand extraembryonic cell types. Pluripotent cells are able to give riseto all embryonic cell types. Multipotent cells include those able togive rise to a subset of cell lineages, but all within a particulartissue, organ, or physiological system (for example, hematopoietic stemcells (HSC) can produce progeny that include HSC (self-renewal), bloodcell-restricted oligopotent progenitors, and all cell types and elements(e.g., platelets) that are normal components of the blood). Cells thatare oligopotent can give rise to a more restricted subset of celllineages than multipotent stem cells; and cells that are unipotent areable to give rise to a single cell lineage (e.g., spermatogenic stemcells).

Stem cells are also categorized on the basis of the source from whichthey may be obtained. An adult stem cell is generally a multipotentundifferentiated cell found in tissue comprising multiple differentiatedcell types. The adult stem cell can renew itself. Under normalcircumstances, it can also differentiate to yield the specialized celltypes of the tissue from which it originated, and possibly other tissuetypes. An embryonic stem cell is a pluripotent cell from the inner cellmass of a blastocyst-stage embryo. A fetal stem cell is one thatoriginates from fetal tissues or membranes. A postpartum stem cell is amultipotent or pluripotent cell that originates substantially fromextraembryonic tissue available after birth, namely, the placenta andthe umbilical cord. These cells have been found to possess featurescharacteristic of pluripotent stem cells, including rapid proliferationand the potential for differentiation into many cell lineages.Postpartum stem cells may be blood-derived (e.g., as are those obtainedfrom umbilical cord blood) or non-blood-derived (e.g., as obtained fromthe non-blood tissues of the umbilical cord and placenta).

Embryonic tissue is typically defined as tissue originating from theembryo (which in humans refers to the period from fertilization to aboutsix weeks of development. Fetal tissue refers to tissue originating fromthe fetus, which in humans refers to the period from about six weeks ofdevelopment to parturition. Extraembryonic tissue is tissue associatedwith, but not originating from, the embryo or fetus. Extraembryonictissues include extraembryonic membranes (chorion, amnion, yolk sac andallantois), umbilical cord and placenta (which itself forms from thechorion and the maternal decidua basalis).

Differentiation is the process by which an unspecialized (“uncommitted”)or less specialized cell acquires the features of a specialized cell,such as a nerve cell or a muscle cell, for example. A differentiatedcell is one that has taken on a more specialized (“committed”) positionwithin the lineage of a cell. The term committed, when applied to theprocess of differentiation, refers to a cell that has proceeded in thedifferentiation pathway to a point where, under normal circumstances, itwill continue to differentiate into a specific cell type or subset ofcell types, and cannot, under normal circumstances, differentiate into adifferent cell type or revert to a less differentiated cell type.De-differentiation refers to the process by which a cell reverts to aless specialized (or committed) position within the lineage of a cell.As used herein, the lineage of a cell defines the heredity of the cell,i.e. which cells it came from and what cells it can give rise to. Thelineage of a cell places the cell within a hereditary scheme ofdevelopment and differentiation.

In a broad sense, a progenitor cell is a cell that has the capacity tocreate progeny that are more differentiated than itself, and yet retainsthe capacity to replenish the pool of progenitors. By that definition,stem cells themselves are also progenitor cells, as are the moreimmediate precursors to terminally differentiated cells. When referringto the cells of the present invention, as described in greater detailbelow, this broad definition of progenitor cell may be used. In anarrower sense, a progenitor cell is often defined as a cell that isintermediate in the differentiation pathway, i.e., it arises from a stemcell and is intermediate in the production of a mature cell type orsubset of cell types. This type of progenitor cell is generally not ableto self-renew. Accordingly, if this type of cell is referred to herein,it will be referred to as a non-renewing progenitor cell or as anintermediate progenitor or precursor cell.

As used herein, the phrase differentiates into a neural lineage orphenotype refers to a cell that becomes partially or fully committed toa specific neural phenotype of the CNS or PNS, i.e., a neuron or a glialcell, the latter category including without limitation astrocytes,oligodendrocytes, Schwann cells and microglia.

The cells of the present invention are generally referred to aspostpartum-derived cells (or PPDCs). They also may sometimes be referredto more specifically as umbilicus-derived cells or placenta-derivedcells (UDCs or PDCs). In addition, the cells may be described as beingstem or progenitor cells, the latter term being used in the broad sense.The term derived is used to indicate that the cells have been obtainedfrom their biological source and grown or otherwise manipulated in vitro(e.g., cultured in a Growth Medium to expand the population and/or toproduce a cell line). The in vitro manipulations of umbilical stem cellsand the unique features of the umbilicus-derived cells of the presentinvention are described in detail below.

Various terms are used to describe cells in culture. Cell culture refersgenerally to cells taken from a living organism and grown undercontrolled condition (“in culture” or “cultured”). A primary cellculture is a culture of cells, tissues, or organs taken directly from anorganism(s) before the first subculture. Cells are expanded in culturewhen they are placed in a Growth Medium under conditions that facilitatecell growth and/or division, resulting in a larger population of thecells. When cells are expanded in culture, the rate of cellproliferation is sometimes measured by the amount of time needed for thecells to double in number. This is referred to as doubling time.

A cell line is a population of cells formed by one or moresubcultivations of a primary cell culture. Each round of subculturing isreferred to as a passage. When cells are subcultured, they are referredto as having been passaged. A specific population of cells, or a cellline, is sometimes referred to or characterized by the number of timesit has been passaged. For example, a cultured cell population that hasbeen passaged ten times may be referred to as a P10 culture. The primaryculture, i.e., the first culture following the isolation of cells fromtissue, is designated P0. Following the first subculture, the cells aredescribed as a secondary culture (P1 or passage 1). After the secondsubculture, the cells become a tertiary culture (P2 or passage 2), andso on. It will be understood by those of skill in the art that there maybe many population doublings during the period of passaging; thereforethe number of population doublings of a culture is greater than thepassage number. The expansion of cells (i.e., the number of populationdoublings) during the period between passaging depends on many factors,including but not limited to the seeding density, substrate, medium,growth conditions, and time between passaging.

A conditioned medium is a medium in which a specific cell or populationof cells has been cultured, and then removed. When cells are cultured ina medium, they may secrete cellular factors that can provide trophicsupport to other cells. Such trophic factors include, but are notlimited to hormones, cytokines, extracellular matrix (ECM), proteins,vesicles, antibodies, and granules. The medium containing the cellularfactors is the conditioned medium.

Generally, a trophic factor is defined as a substance that promotessurvival, growth, differentiation, proliferation and/or maturation of acell, or stimulates increased activity of a cell. The interactionbetween cells via trophic factors may occur between cells of differenttypes. Cell interaction by way of trophic factors is found inessentially all cell types, and is a particularly significant means ofcommunication among neural cell types. Trophic factors also can functionin an autocrine fashion, i.e., a cell may produce trophic factors thataffect its own survival, growth, differentiation, proliferation and/ormaturation.

When referring to cultured vertebrate cells, the term senescence (alsoreplicative senescence or cellular senescence) refers to a propertyattributable to finite cell cultures; namely, their inability to growbeyond a finite number of population doublings (sometimes referred to asHayflick's limit). Although cellular senescence was first describedusing fibroblast-like cells, most normal human cell types that can begrown successfully in culture undergo cellular senescence. The in vitrolifespan of different cell types varies, but the maximum lifespan istypically fewer than 100 population doublings (this is the number ofdoublings for all the cells in the culture to become senescent and thusrender the culture unable to divide). Senescence does not depend onchronological time, but rather is measured by the number of celldivisions, or population doublings, the culture has undergone. Thus,cells made quiescent by removing essential growth factors are able toresume growth and division when the growth factors are re-introduced,and thereafter carry out the same number of doublings as equivalentcells grown continuously. Similarly, when cells are frozen in liquidnitrogen after various numbers of population doublings and then thawedand cultured, they undergo substantially the same number of doublings ascells maintained unfrozen in culture. Senescent cells are not dead ordying cells; they are actually resistant to programmed cell death(apoptosis), and have been maintained in their nondividing state for aslong as three years. These cells are very much alive and metabolicallyactive, but they do not divide. The nondividing state of senescent cellshas not yet been found to be reversible by any biological, chemical, orviral agent.

The term neurodegenerative condition (or disorder) is an inclusive termencompassing acute and chronic conditions, disorders or diseases of thecentral or peripheral nervous system. A neurodegenerative condition maybe age-related, or it may result from injury or trauma, or it may berelated to a specific disease or disorder. Acute neurodegenerativeconditions include, but are not limited to, conditions associated withneuronal cell death or compromise including cerebrovascularinsufficiency, focal or diffuse brain trauma, diffuse brain damage,spinal cord injury or peripheral nerve trauma, e.g., resulting fromphysical or chemical burns, deep cuts or limb severance. Examples ofacute neurodegenerative disorders are: cerebral ischemia or infarctionincluding embolic occlusion and thrombotic occlusion, reperfusionfollowing acute ischemia, perinatal hypoxic-ischemic injury, cardiacarrest, as well as intracranial hemorrhage of any type (such asepidural, subdural, subarachnoid and intracerebral), and intracranialand intravertebral lesions (such as contusion, penetration, shear,compression and laceration), as well as whiplash and shaken infantsyndrome. Chronic neurodegenerative conditions include, but are notlimited to, Alzheimer's disease, Pick's disease, diffuse Lewy bodydisease, progressive supranuclear palsy (Steel-Richardson syndrome),multisystem degeneration (Shy-Drager syndrome), chronic epilepticconditions associated with neurodegeneration, motor neuron diseasesincluding amyotrophic lateral sclerosis, degenerative ataxias, corticalbasal degeneration, ALS-Parkinson's-Dementia complex of Guam, subacutesclerosing panencephalitis, Huntington's disease, Parkinson's disease,synucleinopathies (including multiple system atrophy), primaryprogressive aphasia, striatonigral degeneration, Machado-Josephdisease/spinocerebellar ataxia type 3 and olivopontocerebellardegenerations, Gilles De La Tourette's disease, bulbar and pseudobulbarpalsy, spinal and spinobulbar muscular atrophy (Kennedy's disease),primary lateral sclerosis, familial spastic paraplegia, Werdnig-Hoffmanndisease, Kugelberg-Welander disease, Tay-Sach's disease, Sandhoffdisease, familial spastic disease, Wohlfart-Kugelberg-Welander disease,spastic paraparesis, progressive multifocal leukoencephalopathy,familial dysautonomia (Riley-Day syndrome), and prion diseases(including, but not limited to Creutzfeldt-Jakob,Gerstmann-Sträussler-Scheinker disease, Kuru and fatal familialinsomnia), demyelination diseases and disorders including multiplesclerosis and hereditary diseases such as leukodystrophies.

Other neurodegenerative conditions include tumors and other neoplasticconditions affecting the CNS and PNS. Though the underlying disease isconsidered proliferative (rather than neurodegenerative), surroundingtissues may be compromised. Furthermore, cell therapy may be utilized todeliver apoptotic or other antineoplastic molecules to the tumor site,e.g., via delivery of genetically modified cells producing such agents.

Other neurodegenerative conditions include various neuropathies, such asmultifocal neuropathies, sensory neuropathies, motor neuropathies,sensory-motor neuropathies, infection-related neuropathies, autonomicneuropathies, sensory-autonomic neuropathies, demyelinating neuropathies(including, but not limited to, Guillain-Barre syndrome and chronicinflammatory demyelinating polyradiculoneuropathy), other inflammatoryand immune neuropathies, neuropathies induced by drugs, neuropathiesinduced by pharmacological treatments, neuropathies induced by toxins,traumatic neuropathies (including, but not limited to, compression,crush, laceration and segmentation neuropathies), metabolicneuropathies, endocrine and paraneoplastic neuropathies, among others.

Other neurodegenerative conditions include dementias, regardless ofunderlying etiology, including age-related dementia and other dementiasand conditions with memory loss including dementia associated withAlzheimer's disease, vascular dementia, diffuse white matter disease(Binswanger's disease), dementia of endocrine or metabolic origin,dementia of head trauma and diffuse brain damage, dementia pugilisticaand frontal lobe dementia.

The term treating (or treatment of) a neurodegenerative condition refersto ameliorating the effects of, or delaying, halting or reversing theprogress of, or delaying or preventing the onset of, a neurodegenerativecondition as defined herein.

The term effective amount refers to a concentration or amount of areagent or pharmaceutical composition, such as a growth factor,differentiation agent, trophic factor, cell population or other agent,that is effective for producing an intended result, including cellgrowth and/or differentiation in vitro or in vivo, or treatment of aneurodegenerative condition as described herein. With respect to growthfactors, an effective amount may range from about 1 nanogram/milliliterto about 1 microgram/milliliter. With respect to PPDCs as administeredto a patient in vivo, an effective amount may range from as few asseveral hundred or fewer to as many as several million or more. Inspecific embodiments, an effective amount may range from 10³-10¹¹, morespecifically at least about 10⁴ cells. It will be appreciated that thenumber of cells to be administered will vary depending on the specificsof the disorder to be treated, including but not limited to size ortotal volume/surface area to be treated, as well as proximity of thesite of administration to the location of the region to be treated,among other factors familiar to the medicinal biologist.

The terms effective period (or time) and effective conditions refer to aperiod of time or other controllable conditions (e.g., temperature,humidity for in vitro methods), necessary or preferred for an agent orpharmaceutical composition to achieve its intended result.

The term patient or subject refers to animals, including mammals,preferably humans, who are treated with the pharmaceutical compositionsor in accordance with the methods described herein.

The term pharmaceutically acceptable carrier (or medium), which may beused interchangeably with the term biologically compatible carrier ormedium, refers to reagents, cells, compounds, materials, compositions,and/or dosage forms that are not only compatible with the cells andother agents to be administered therapeutically, but also are, withinthe scope of sound medical judgment, suitable for use in contact withthe tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other complication commensurate with areasonable benefit/risk ratio. As described in greater detail herein,pharmaceutically acceptable carriers suitable for use in the presentinvention include liquids, semi-solid (e.g., gels) and solid materials(e.g., cell scaffolds and matrices, tubes sheets and other suchmaterials as known in the art and described in greater detail herein).These semi-solid and solid materials may be designed to resistdegradation within the body (non-biodegradable) or they may be designedto degrade within the body (biodegradable, bioerodable). A biodegradablematerial may further be bioresorbable or bioabsorbable, i.e., it may bedissolved and absorbed into bodily fluids (water-soluble implants areone example), or degraded and ultimately eliminated from the body,either by conversion into other materials or breakdown and eliminationthrough natural pathways.

Several terms are used herein with respect to cell replacement therapy.The terms autologous transfer, autologous transplantation, autograft andthe like refer to treatments wherein the cell donor is also therecipient of the cell replacement therapy. The terms allogeneictransfer, allogeneic transplantation, allograft and the like refer totreatments wherein the cell donor is of the same species as therecipient of the cell replacement therapy, but is not the sameindividual. A cell transfer in which the donor's cells and have beenhistocompatibly matched with a recipient is sometimes referred to as asyngeneic transfer. The terms xenogeneic transfer, xenogeneictransplantation, xenograft and the like refer to treatments wherein thecell donor is of a different species than the recipient of the cellreplacement therapy.

Description

Neurodegenerative conditions, which encompass acute, chronic andprogressive disorders and diseases having widely divergent causes, haveas a common feature the dysfunction or loss of a specific or vulnerablegroup of neural cells. This commonality enables development of similartherapeutic approaches for the repair and regeneration of vulnerable ordamaged neural tissue, one of which is cell-based therapy. In itsvarious embodiments described herein, the present invention featuresmethods and pharmaceutical compositions for neural repair andregeneration that utilize progenitor cells and cell populations derivedfrom postpartum tissues. The invention is applicable to anyneurodegenerative condition, but is expected to be particularly suitablefor a number of neural disorders for which treatment or cure heretoforehas been difficult or unavailable. These include, without limitation,Parkinson's disease, Alzheimer's disease, Huntington's disease, stroke,amyotrophic lateral sclerosis, multiple sclerosis, spinal cord injuryand peripheral nerve injury (e.g., as associated with diabeticneuropathy).

As summarized above, the invention, in one of its aspects is generallydirected to isolated postpartum-derived cells (PPDCs), which are derivedfrom placental or umbilical cord tissue that has been renderedsubstantially free of blood. The PPDCs are capable of self-renewal andexpansion in culture and have the potential to differentiate into cellsof neural phenotypes. Certain embodiments features populationscomprising such cells, pharmaceutical compositions comprising the cellsor components or products thereof, and methods of using thepharmaceutical compositions for treatment of patients with acute orchronic neurodegenerative conditions. The postpartum-derived cells havebeen characterized by their growth properties in culture, by their cellsurface markers, by their gene expression, by their ability to producecertain biochemical trophic factors, and by their immunologicalproperties.

Preparation of PPDCs

According to the methods described herein, a mammalian placenta andumbilical cord are recovered upon or shortly after termination of eithera full-term or pre-term pregnancy, for example, after expulsion afterbirth. The postpartum tissue may be transported from the birth site to alaboratory in a sterile container such as a flask, beaker, culture dish,or bag. The container may have a solution or medium, including but notlimited to a salt solution, such as, for example, Dulbecco's ModifiedEagle's Medium (DMEM) or phosphate buffered saline (PBS), or anysolution used for transportation of organs used for transplantation,such as University of Wisconsin solution or perfluorochemical solution.One or more antibiotic and/or antimycotic agents, such as but notlimited to penicillin, streptomycin, amphotericin B, gentamicin, andnystatin, may be added to the medium or buffer. The postpartum tissuemay be rinsed with an anticoagulant solution such as heparin-containingsolution. It is preferable to keep the tissue at about 4-10° C. prior toextraction of PPDCs. It is even more preferable that the tissue not befrozen prior to extraction of PPDCs.

Isolation of PPDCs preferably occurs in an aseptic environment. Theumbilical cord may be separated from the placenta by means known in theart. Alternatively, the umbilical cord and placenta are used withoutseparation. Blood and debris are preferably removed from the postpartumtissue prior to isolation of PPDCs. For example, the postpartum tissuemay be washed with buffer solution, such as but not limited to phosphatebuffered saline. The wash buffer also may comprise one or moreantimycotic and/or antibiotic agents, such as but not limited topenicillin, streptomycin, amphotericin B, gentamicin, and nystatin.

Postpartum tissue comprising a whole placenta or a fragment or sectionthereof is disaggregated by mechanical force (mincing or shear forces).In a presently preferred embodiment, the isolation procedure alsoutilizes an enzymatic digestion process. Many enzymes are known in theart to be useful for the isolation of individual cells from complextissue matrices to facilitate growth in culture. Ranging from weaklydigestive (e.g. deoxyribonucleases and the neutral protease, dispase) tostrongly digestive (e.g. papain and trypsin), such enzymes are availablecommercially. A nonexhaustive list of enzymes compatible herewithincludes mucolytic enzyme activities, metalloproteases, neutralproteases, serine proteases (such as trypsin, chymotrypsin, orelastase), and deoxyribonucleases. Presently preferred are enzymeactivities selected from metalloproteases, neutral proteases andmucolytic activities. For example, collagenases are known to be usefulfor isolating various cells from tissues. Deoxyribonucleases can digestsingle-stranded DNA and can minimize cell-clumping during isolation.Preferred methods involve enzymatic treatment with for examplecollagenase and dispase, or collagenase, dispase, and hyaluronidase, andsuch methods are provided wherein in certain preferred embodiments, amixture of collagenase and the neutral protease dispase are used in thedissociating step. More preferred are those methods which employdigestion in the presence of at least one collagenase from Clostridiumhistolyticum, and either of the protease activities, dispase andthermolysin. Still more preferred are methods employing digestion withboth collagenase and dispase enzyme activities. Also preferred aremethods which include digestion with a hyaluronidase activity inaddition to collagenase and dispase activities. The skilled artisan willappreciate that many such enzyme treatments are known in the art forisolating cells from various tissue sources. For example, the LIBERASEBlendzyme (Roche) series of enzyme combinations are suitable for use inthe instant methods. Other sources of enzymes are known, and the skilledartisan may also obtain such enzymes directly from their naturalsources. The skilled artisan is also well-equipped to assess new, oradditional enzymes or enzyme combinations for their utility in isolatingthe cells of the invention. Preferred enzyme treatments are 0.5, 1, 1.5,or 2 hours long or longer. In other preferred embodiments, the tissue isincubated at 37° C. during the enzyme treatment of the dissociationstep.

In some embodiments of the invention, postpartum tissue is separatedinto sections comprising various aspects of the tissue, such asneonatal, neonatal/maternal, and maternal aspects of the placenta, forinstance. The separated sections then are dissociated by mechanicaland/or enzymatic dissociation according to the methods described herein.Cells of neonatal or maternal lineage may be identified by any meansknown in the art, for example, by karyotype analysis or in situhybridization for a Y chromosome.

Isolated cells or postpartum tissue from which PPDCs grow out may beused to initiate, or seed, cell cultures. Isolated cells are transferredto sterile tissue culture vessels either uncoated or coated withextracellular matrix or ligands such as laminin, collagen (native,denatured or crosslinked), gelatin, fibronectin, and other extracellularmatrix proteins. PPDCs are cultured in any culture medium capable ofsustaining growth of the cells such as, but not limited to, DMEM (highor low glucose), advanced DMEM, DMEM/MCDB 201, Eagle's basal medium,Ham's F10 medium (F10), Ham's F-12 medium (F12), Iscove's modifiedDulbecco's medium, Mesenchymal Stem Cell Growth Medium (MSCGM),DMEM/F12, RPMI 1640, and CELL-GRO—FREE. The culture medium may besupplemented with one or more components including, for example, fetalbovine serum (FBS), preferably about 2-15% (v/v); equine serum (ES);human serum(HS); beta-mercaptoethanol (BME or 2-ME), preferably about0.001% (v/v); one or more growth factors, for example, platelet-derivedgrowth factor (PDGF), epidermal growth factor (EGF), fibroblast growthfactor (FGF), vascular endothelial growth factor (VEGF), insulin-likegrowth factor-1 (IGF-1), leukocyte inhibitory factor (LIF) anderythropoietin; amino acids, including L-valine; and one or moreantibiotic and/or antimycotic agents to control microbial contamination,such as, for example, penicillin G, streptomycin sulfate, amphotericinB, gentamicin, and nystatin, either alone or in combination. The culturemedium preferably comprises Growth Medium (DMEM-low glucose, serum, BME,and an antibiotic agent).

The cells are seeded in culture vessels at a density to allow cellgrowth. In a preferred embodiment, the cells are cultured at about 0 toabout 5 percent by volume CO₂ in air. In some preferred embodiments, thecells are cultured at about 2 to about 25 percent O₂ in air, preferablyabout 5 to about 20 percent O₂ in air. The cells preferably are culturedat about 25 to about 40° C. and more preferably are cultured at 37° C.The cells are preferably cultured in an incubator. The medium in theculture vessel can be static or agitated, for example, using abioreactor. PPDCs preferably are grown under low oxidative stress (e.g.,with addition of glutathione, Vitamin C, Catalase, Vitamin E,N-Acetylcysteine). “Low oxidative stress”, as used herein, refers toconditions of no or minimal free radical damage to the cultured cells.

Methods for the selection of the most appropriate culture medium, mediumpreparation, and cell culture techniques are well known in the art andare described in a variety of sources, including Doyle et al., (eds.),1995, CELL & TISSUE CULTURE: LABORATORY PROCEDURES, John Wiley & Sons,Chichester; and Ho and Wang (eds.), 1991, ANIMAL CELL BIOREACTORS,Butterworth-Heinemann, Boston, which are incorporated herein byreference.

After culturing the isolated cells or tissue fragments for a sufficientperiod of time, PPDCs will have grown out, either as a result ofmigration from the postpartum tissue or cell division, or both. In someembodiments of the invention, PPDCs are passaged, or removed to aseparate culture vessel containing fresh medium of the same or adifferent type as that used initially, where the population of cells canbe mitotically expanded. The cells of the invention may be used at anypoint between passage 0 and senescence. The cells preferably arepassaged between about 3 and about 25 times, more preferably arepassaged about 4 to about 12 times, and preferably are passaged 10 or 11times. Cloning and/or subcloning may be performed to confirm that aclonal population of cells has been isolated.

In some aspects of the invention, the different cell types present inpostpartum tissue are fractionated into subpopulations from which thePPDCs can be isolated. This may be accomplished using standardtechniques for cell separation including, but not limited to, enzymatictreatment to dissociate postpartum tissue into its component cells,followed by cloning and selection of specific cell types, for examplebut not limited to selection based on morphological and/or biochemicalmarkers; selective growth of desired cells (positive selection),selective destruction of unwanted cells (negative selection); separationbased upon differential cell agglutinability in the mixed population as,for example, with soybean agglutinin; freeze-thaw procedures;differential adherence properties of the cells in the mixed population;filtration; conventional and zonal centrifugation; centrifugalelutriation (counter-streaming centrifugation); unit gravity separation;countercurrent distribution; electrophoresis; and fluorescence activatedcell sorting (FACS). For a review of clonal selection and cellseparation techniques, see Freshney, 1994, CULTURE OF ANIMAL CELLS: AMANUAL OF BASIC TECHNIQUES, 3rd Ed., Wiley-Liss, Inc., New York, whichis incorporated herein by reference.

The culture medium is changed as necessary, for example, by carefullyaspirating the medium from the dish, for example, with a pipette, andreplenishing with fresh medium. Incubation is continued until asufficient number or density of cells accumulate in the dish. Theoriginal explanted tissue sections may be removed and the remainingcells trypsinized using standard techniques or using a cell scraper.After trypsinization, the cells are collected, removed to fresh mediumand incubated as above. In some embodiments, the medium is changed atleast once at approximately 24 hours post-trypsinization to remove anyfloating cells. The cells remaining in culture are considered to bePPDCs.

PPDCs may be cryopreserved. Accordingly, in a preferred embodimentdescribed in greater detail below, PPDCs for autologous transfer (foreither the mother or child) may be derived from appropriate postpartumtissues following the birth of a child, then cryopreserved so as to beavailable in the event they are later needed for transplantation.

Characteristics of PPDCs

PPDCs may be characterized, for example, by growth characteristics(e.g., population doubling capability, doubling time, passages tosenescence), karyotype analysis (e.g., normal karyotype; maternal orneonatal lineage), flow cytometry (e.g., FACS analysis),immunohistochemistry and/or immunocytochemistry (e.g., for detection ofepitopes), gene expression profiling (e.g., gene chip arrays; polymerasechain reaction (for example, reverse transcriptase PCR, real time PCR,and conventional PCR)), protein arrays, protein secretion (e.g., byplasma clotting assay or analysis of PDC-conditioned medium, forexample, by Enzyme Linked ImmunoSorbent Assay (ELISA)), mixed lymphocytereaction (e.g., as measure of stimulation of PBMCs), and/or othermethods known in the art.

Examples of PPDCs derived from placental tissue were deposited with theAmerican Type Culture Collection (ATCC, Manassas, Va.) and assigned ATCCAccession Numbers as follows: (1) strain designation PLA 071003 (P8) wasdeposited Jun. 15, 2004 and assigned Accession No. PTA-6074; (2) straindesignation PLA 071003 (P11) was deposited Jun. 15, 2004 and assignedAccession No. PTA-6075; and (3) strain designation PLA 071003 (P16) wasdeposited Jun. 16, 2004 and assigned Accession No. PTA-6079. Examples ofPPDCs derived from umbilicus tissue were deposited with the AmericanType Culture Collection on Jun. 10, 2004, and assigned ATCC AccessionNumbers as follows: (1) strain designation UMB 022803 (P7) was assignedAccession No. PTA-6067; and (2) strain designation UMB 022803 (P17) wasassigned Accession No. PTA-6068.

In various embodiments, the PPDCs possess one or more of the followinggrowth features (1) they require L-valine for growth in culture; (2)they are capable of growth in atmospheres containing oxygen from about5% to at least about 20% (3) they have the potential for at least about40 doublings in culture before reaching senescence; and (4) they attachand expand on a coated or uncoated tissue culture vessel, wherein thecoated tissue culture vessel comprises a coating of gelatin, laminin,collagen, polyornithine, vitronectin or fibronectin.

In certain embodiments the PPDCs possess a normal karyotype, which ismaintained as the cells are passaged. Karyotyping is particularly usefulfor identifying and distinguishing neonatal from maternal cells derivedfrom placenta. Methods for karyotyping are available and known to thoseof skill in the art.

In other embodiments, the PPDCs may be characterized by production ofcertain proteins, including (1) production of at least one of tissuefactor, vimentin, and alpha-smooth muscle actin; and (2) production ofat least one of CD10, CD13, CD44, CD73, CD90, PDGFr-alpha, PD-L2 andHLA-A,B,C cell surface markers, as detected by flow cytometry. In otherembodiments, the PPDCs may be characterized by lack of production of atleast one of CD31, CD34, CD45, CD80, CD86, CD117, CD141, CD178, B7-H2,HLA-G, and HLA-DR,DP,DQ cell surface markers, as detected by flowcytometry. Particularly preferred are cells that produce at least two oftissue factor, vimentin, and alpha-smooth muscle actin. More preferredare those cells producing all three of the proteins tissue factor,vimentin, and alpha-smooth muscle actin.

In other embodiments, the PPDCs may be characterized by gene expression,which relative to a human cell that is a fibroblast, a mesenchymal stemcell, or an iliac crest bone marrow cell, is increased for a geneencoding at least one of interleukin 8; reticulon 1; chemokine (C-X-Cmotif) ligand 1 (melonoma growth stimulating activity, alpha); chemokine(C-X-C motif) ligand 6 (granulocyte chemotactic protein 2); chemokine(C-X-C motif) ligand 3; tumor necrosis factor, alpha-induced protein 3;C-type lectin superfamily member 2; Wilms tumor 1; aldehydedehydrogenase 1 family member A2; renin; oxidized low densitylipoprotein receptor 1; Homo sapiens clone IMAGE:4179671; protein kinaseC zeta; hypothetical protein DKFZp564F013; downregulated in ovariancancer 1; and Homo sapiens gene from clone DKFZp547k1113.

In yet other embodiments, the PPDCs may be characterized by geneexpression, which relative to a human cell that is a fibroblast, amesenchymal stem cell, or an iliac crest bone marrow cell, is reducedfor a gene encoding at least one of: short stature homeobox 2; heatshock 27 kDa protein 2; chemokine (C-X-C motif) ligand 12 (stromalcell-derived factor 1); elastin (supravalvular aortic stenosis,Williams-Beuren syndrome); Homo sapiens mRNA; cDNA DKFZp586M2022 (fromclone DKFZp586M2022); mesenchyme homeo box 2 (growth arrest-specifichomeo box); sine oculis homeobox homolog 1 (Drosophila); crystallin,alpha B; disheveled associated activator of morphogenesis 2;DKFZP586B2420 protein; similar to neuralin 1; tetranectin (plasminogenbinding protein); src homology three (SH3) and cysteine rich domain;cholesterol 25-hydroxylase; runt-related transcription factor 3;interleukin 11 receptor, alpha; procollagen C-endopeptidase enhancer;frizzled homolog 7 (Drosophila); hypothetical gene BC008967; collagen,type VIII, alpha 1; tenascin C (hexabrachion); iroquois homeobox protein5; hephaestin; integrin, beta 8; synaptic vesicle glycoprotein 2;neuroblastoma, suppression of tumorigenicity 1; insulin-like growthfactor binding protein 2, 36 kDa; Homo sapiens cDNA FLJ12280 fis, cloneMAMMA1001744; cytokine receptor-like factor 1; potassiumintermediate/small conductance calcium-activated channel, subfamily N,member 4; integrin, beta 7; transcriptional co-activator withPDZ-binding motif (TAZ); sine oculis homeobox homolog 2 (Drosophila);KIAA1034 protein; vesicle-associated membrane protein 5 (myobrevin);EGF-containing fibulin-like extracellular matrix protein 1; early growthresponse 3; distal-less homeo box 5; hypothetical protein FLJ20373;aldo-keto reductase family 1, member C3 (3-alpha hydroxysteroiddehydrogenase, type II); biglycan; transcriptional co-activator withPDZ-binding motif (TAZ); fibronectin 1; proenkephalin; integrin,beta-like 1 (with EGF-like repeat domains); Homo sapiens mRNA fulllength insert cDNA clone EUROIMAGE 1968422; EphA3; KIAA0367 protein;natriuretic peptide receptor C/guanylate cyclase C (atrionatriureticpeptide receptor C); hypothetical protein FLJ14054; Homo sapiens mRNA;cDNA DKFZp564B222 (from clone DKFZp564B222); BCL2/adenovirus E1B 19 kDainteracting protein 3-like; AE binding protein 1; and cytochrome coxidase subunit VIla polypeptide 1 (muscle).

In other embodiments, the PPDCs may be characterized by secretion of atleast one of MCP-1, IL-6, IL-8, GCP-2, HGF, KGF, FGF, HB-EGF, BDNF, TPO,MIP1a, RANTES, and TIMP1. In alternative embodiments, the PPDCs may becharacterized by lack of secretion of at least one of TGF-beta2, ANG2,PDGFbb, MIP1b, I309, MDC, and VEGF, as detected by ELISA.

In preferred embodiments, the cell comprises two or more of theabove-listed growth, protein/surface marker production, gene expressionor substance-secretion characteristics. More preferred are those cellscomprising, three, four, or five or more of the characteristics. Stillmore preferred are PPDCs comprising six, seven, or eight or more of thecharacteristics. Still more preferred presently are those cellscomprising all of above characteristics.

Among cells that are presently preferred for use with the invention inseveral of its aspects are postpartum cells having the characteristicsdescribed above and more particularly those wherein the cells havenormal karyotypes and maintain normal karyotypes with passaging, andfurther wherein the cells express each of the markers CD10, CD13, CD44,CD73, CD90, PDGFr-alpha, and HLA-A,B,C, wherein the cells produce theimmunologically-detectable proteins which correspond to the listedmarkers. Still more preferred are those cells which in addition to theforegoing do not produce proteins corresponding to any of the markersCD31, CD34, CD45, CD117, CD141, or HLA-DR,DP,DQ, as detected by flowcytometry.

Certain cells having the potential to differentiate along lines leadingto various phenotypes are unstable and thus can spontaneouslydifferentiate. Presently preferred for use with the invention are cellsthat do not spontaneously differentiate, for example along neural lines.Preferred cells, when grown in Growth Medium, are substantially stablewith respect to the cell markers produced on their surface, and withrespect to the expression pattern of various genes, for example asdetermined using an Affymetrix GENECHIP. The cells remain substantiallyconstant, for example in their surface marker characterisitics overpassaging, through multiple population doublings.

However, one feature of PPDCs is that they may be deliberately inducedto differentiate into neural lineage phenotypes by subjecting them todifferentiation-inducing cell culture conditions. This may beaccomplished by one or more methods known in the art. For instance, asexemplified herein, PPDCs may be plated on flasks coated with laminin inNeurobasal-A medium (Invitrogen, Carlsbad, Calif.) containing B27 (B27supplement, Invitrogen), L-glutamine and Penicillin/Streptomycin, thecombination of which is referred to herein as Neural ProgenitorExpansion (NPE) medium. NPE media may be further supplemented with bFGFand/or EGF. Alternatively, PPDCs may be induced to differentiate invitro by (1) co-culturing the PPDCs with neural progenitor cells, or (2)growing the PPDCs in neural progenitor cell-conditioned medium.

Differentiation of the PPDCs may be demonstrated by a bipolar cellmorphology with extended processes. The induced cell populations maystain positive for the presence of nestin. Differentiated PPDCs may beassessed by detection of nestin, TuJ1 (BIII tubulin), GFAP, tyrosinehydroxylase, GABA, O4 and/or MBP. In some embodiments, PPDCs haveexhibited the ability to form three-dimensional bodies characteristic ofneuronal stem cell formation of neurospheres.

PPDC Populations, Modifications, Components and Products

Another aspect of the invention features populations of the PPDCsdescribed above. In some embodiments, the cell population isheterogeneous. A heterogeneous cell population of the invention maycomprise at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,or 95% PPDCs of the invention. The heterogeneous cell populations of theinvention may further comprise stem cells or other progenitor cells,such as neural progenitor cells, or it may further comprise fullydifferentiated neural cells. In some embodiments, the population issubstantially homogeneous, i.e., comprises substantially only PPDCs(preferably at least about 96%, 97%, 98%, 99% or more PPDCs). Thehomogeneous cell population of the invention may comprise umbilicus- orplacenta-derived cells. Homogeneous populations of umbilicus-derivedcells are preferably free of cells of maternal lineage. Homogeneouspopulations of placenta-derived cells may be of neonatal or maternallineage. Homogeneity of a cell population may be achieved by any methodknown in the art, for example, by cell sorting (e.g., flow cytometry) orby clonal expansion in accordance with known methods. Thus, preferredhomogeneous PPDC populations may comprise a clonal cell line ofpostpartum-derived cells. Such populations are particularly useful whena cell clone with highly desirable functionality has been isolated.

Also provided herein are populations of cells incubated in the presenceof one or more factors, or under conditions, that stimulate stem celldifferentiation along a neurogenic pathway. Such factors are known inthe art and the skilled artisan will appreciate that determination ofsuitable conditions for differentiation can be accomplished with routineexperimentation. Optimization of such conditions can be accomplished bystatistical experimental design and analysis, for example responsesurface methodology allows simultaneous optimization of multiplevariables, for example in a biological culture. Presently preferredfactors include, but are not limited to factors, such as growth ortrophic factors, demethylating agents, co-culture with neural lineagecells or culture in neural lineage cell-conditioned medium, as wellother conditions known in the art to stimulate stem cell differentiationalong a neurogenic pathway or lineage (see, e.g., Lang, K. J. D. et al.,2004, J. Neurosci. Res. 76: 184-192; Johe, K. K. et al., 1996, GenesDevel. 10: 3129-3140; Gottleib, D., 2002, Ann. Rev. Neurosci. 25:381-407).

PPDCs may also be genetically modified to produce neurotherapeuticallyuseful gene products, or to produce antineoplastic agents for treatmentof tumors, for example. Genetic modification may be accomplished usingany of a variety of vectors including, but not limited to, integratingviral vectors, e.g., retrovirus vector or adeno-associated viralvectors; non-integrating replicating vectors, e.g., papilloma virusvectors, SV40 vectors, adenoviral vectors; or replication-defectiveviral vectors. Other methods of introducing DNA into cells include theuse of liposomes, electroporation, a particle gun, or by direct DNAinjection.

Hosts cells are preferably transformed or transfected with DNAcontrolled by or in operative association with, one or more appropriateexpression control elements such as promoter or enhancer sequences,transcription terminators, polyadenylation sites, among others, and aselectable marker. Any promoter may be used to drive the expression ofthe inserted gene. For example, viral promoters include, but are notlimited to, the CMV promoter/enhancer, SV 40, papillomavirus,Epstein-Barr virus or elastin gene promoter. In some embodiments, thecontrol elements used to control expression of the gene of interest canallow for the regulated expression of the gene so that the product issynthesized only when needed in vivo. If transient expression isdesired, constitutive promoters are preferably used in a non-integratingand/or replication-defective vector. Alternatively, inducible promoterscould be used to drive the expression of the inserted gene whennecessary. Inducible promoters include, but are not limited to, thoseassociated with metallothionein and heat shock proteins.

Following the introduction of the foreign DNA, engineered cells may beallowed to grow in enriched media and then switched to selective media.The selectable marker in the foreign DNA confers resistance to theselection and allows cells to stably integrate the foreign DNA as, forexample, on a plasmid, into their chromosomes and grow to form fociwhich, in turn, can be cloned and expanded into cell lines. This methodcan be advantageously used to engineer cell lines that express the geneproduct.

The cells of the invention may be genetically engineered to “knock out”or “knock down” expression of factors that promote inflammation orrejection at the implant site. Negative modulatory techniques for thereduction of target gene expression levels or target gene productactivity levels are discussed below. “Negative modulation,” as usedherein, refers to a reduction in the level and/or activity of targetgene product relative to the level and/or activity of the target geneproduct in the absence of the modulatory treatment. The expression of agene native to a neuron or glial cell can be reduced or knocked outusing a number of techniques including, for example, inhibition ofexpression by inactivating the gene using the homologous recombinationtechnique. Typically, an exon encoding an important region of theprotein (or an exon 5′ to that region) is interrupted by a positiveselectable marker, e.g., neo, preventing the production of normal mRNAfrom the target gene and resulting in inactivation of the gene. A genemay also be inactivated by creating a deletion in part of a gene, or bydeleting the entire gene. By using a construct with two regions ofhomology to the target gene that are far apart in the genome, thesequences intervening the two regions can be deleted (Mombaerts et al.,1991, Proc. Nat. Acad. Sci. U.S.A. 88:3084). Antisense, DNAzymes,ribozymes, small interfering RNA (siRNA) and other such molecules thatinhibit expression of the target gene can also be used to reduce thelevel of target gene activity. For example, antisense RNA molecules thatinhibit the expression of major histocompatibility gene complexes (HLA)have been shown to be most versatile with respect to immune responses.Still further, triple helix molecules can be utilized in reducing thelevel of target gene activity. These techniques are described in detailby L. G. Davis et al. (eds), 1994, BASIC METHODS IN MOLECULAR BIOLOGY,2nd ed., Appleton & Lange, Norwalk, Conn.

In other aspects, the invention provides cell lysates and cell solublefractions prepared from PPDCs, or heterogeneous or homogeneous cellpopulations comprising PPDCs, as well as PPDCs or populations thereofthat have been genetically modified or that have been stimulated todifferentiate along a neurogenic pathway. Such lysates and fractionsthereof have many utilities. Use of the PPDC lysate soluble fraction(i.e., substantially free of membranes) in vivo, for example, allows thebeneficial intracellular milieu to be used allogeneically in a patientwithout introducing an appreciable amount of the cell surface proteinsmost likely to trigger rejection, or other adverse immunologicalresponses. Methods of lysing cells are well-known in the art and includevarious means of mechanical disruption, enzymatic disruption, orchemical disruption, or combinations thereof. Such cell lysates may beprepared from cells directly in their Growth Medium and thus containingsecreted growth factors and the like, or may be prepared from cellswashed free of medium in, for example, PBS or other solution. Washedcells may be resuspended at concentrations greater than the originalpopulation density if preferred.

In one embodiment, whole cell lysates are prepared, e.g., by disruptingcells without subsequent separation of cell fractions. In anotherembodiment, a cell membrane fraction is separated from a solublefraction of the cells by routine methods known in the art, e.g.,centrifugation, filtration, or similar methods.

Cell lysates or cell soluble fractions prepared from populations ofpostpartum-derived cells may be used as is, further concentrated, by forexample, ultrafiltration or lyophilization, or even dried, partiallypurified, combined with pharmaceutically-acceptable carriers or diluentsas are known in the art, or combined with other compounds such asbiologicals, for example pharmaceutically useful protein compositions.Cell lysates or fractions thereof may be used in vitro or in vivo, aloneor for example, with autologous or syngeneic live cells. The lysates, ifintroduced in vivo, may be introduced locally at a site of treatment, orremotely to provide, for example needed cellular growth factors to apatient.

In a further embodiment, PPDCs can be cultured in vitro to producebiological products in high yield. For example, such cells, which eithernaturally produce a particular biological product of interest (e.g., atrophic factor), or have been genetically engineered to produce abiological product, can be clonally expanded using the culturetechniques described herein. Alternatively, cells may be expanded in amedium that induces differentiation to a neural lineage. In either case,biological products produced by the cell and secreted into the mediumcan be readily isolated from the conditioned medium using standardseparation techniques, e.g., such as differential protein precipitation,ion-exchange chromatography, gel filtration chromatography,electrophoresis, and HPLC, to name a few. A “bioreactor” may be used totake advantage of the flow method for feeding, for example, athree-dimensional culture in vitro. Essentially, as fresh media ispassed through the three-dimensional culture, the biological product iswashed out of the culture and may then be isolated from the outflow, asabove.

Alternatively, a biological product of interest may remain within thecell and, thus, its collection may require that the cells be lysed, asdescribed above. The biological product may then be purified using anyone or more of the above-listed techniques.

In other embodiments, the invention provides conditioned medium fromcultured PPDCs for use in vitro and in vivo as described below. Use ofthe PPDC conditioned medium allows the beneficial trophic factorssecreted by the PPDCs to be used allogeneically in a patient withoutintroducing intact cells that could trigger rejection, or other adverseimmunological responses. Conditioned medium is prepared by culturingcells in a culture medium, then removing the cells from the medium.

Conditioned medium prepared from populations of postpartum-derived cellsmay be used as is, further concentrated, by for example, ultrafiltrationor lyophilization, or even dried, partially purified, combined withpharmaceutically-acceptable carriers or diluents as are known in theart, or combined with other compounds such as biologicals, for examplepharmaceutically useful protein compositions. Conditioned medium may beused in vitro or in vivo, alone or for example, with autologous orsyngeneic live cells. The conditioned medium, if introduced in vivo, maybe introduced locally at a site of treatment, or remotely to provide,for example needed cellular growth or trophic factors to a patient.

In another embodiment, an extracellular matrix (ECM) produced byculturing PPDCs on liquid, solid or semi-solid substrates is prepared,collected and utilized as an alternative to implanting live cells into asubject in need of tissue repair or replacement. PPDCs are cultured invitro, on a three dimensional framework as described elsewhere herein,under conditions such that a desired amount of ECM is secreted onto theframework. The comprising the new tissue are removed, and the ECMprocessed for further use, for example, as an injectable preparation. Toaccomplish this, cells on the framework are killed and any cellulardebris removed from the framework. This process may be carried out in anumber of different ways. For example, the living tissue can beflash-frozen in liquid nitrogen without a cryopreservative, or thetissue can be immersed in sterile distilled water so that the cellsburst in response to osmotic pressure.

Once the cells have been killed, the cellular membranes may be disruptedand cellular debris removed by treatment with a mild detergent rinse,such as EDTA, CHAPS or a zwitterionic detergent. Alternatively, thetissue can be enzymatically digested and/or extracted with reagents thatbreak down cellular membranes and allow removal of cell contents.Example of such enzymes include, but are not limited to, hyaluronidase,dispase, proteases, and nucleases. Examples of detergents includenon-ionic detergents such as, for example, alkylaryl polyether alcohol(TRITON X-100), octylphenoxy polyethoxy-ethanol (Rohm and HaasPhiladelphia, Pa.), BRIJ-35, a polyethoxyethanol lauryl ether (AtlasChemical Co., San Diego, Calif.), polysorbate 20 (TWEEN 20), apolyethoxyethanol sorbitan monolaureate (Rohm and Haas), polyethylenelauryl ether (Rohm and Haas); and ionic detergents such as, for example,sodium dodecyl sulphate, sulfated higher aliphatic alcohols, sulfonatedalkanes and sulfonated alkylarenes containing 7 to 22 carbon atoms in abranched or unbranched chain.

The collection of the ECM can be accomplished in a variety of ways,depending, for example, on whether the new tissue has been formed on athree-dimensional framework that is biodegradable or non-biodegradable.For example, if the framework is non-biodegradable, the ECM can beremoved by subjecting the framework to sonication, high pressure waterjets, mechanical scraping, or mild treatment with detergents or enzymes,or any combination of the above.

If the framework is biodegradable, the ECM can be collected, forexample, by allowing the framework to degrade or dissolve in solution.Alternatively, if the biodegradable framework is composed of a materialthat can itself be injected along with the ECM, the framework and theECM can be processed in toto for subsequent injection. Alternatively,the ECM can be removed from the biodegradable framework by any of themethods described above for collection of ECM from a non-biodegradableframework. All collection processes are preferably designed so as not todenature the ECM.

After it has been collected, the ECM may be processed further. Forexample, the ECM can be homogenized to fine particles using techniqueswell known in the art such as by sonication, so that it can pass througha surgical needle. The components of the ECM can be crosslinked, ifdesired, by gamma irradiation. Preferably, the ECM can be irradiatedbetween 0.25 to 2 mega rads to sterilize and crosslink the ECM. Chemicalcrosslinking using agents that are toxic, such as glutaraldehyde, ispossible but not generally preferred.

The amounts and/or ratios of proteins, such as the various types ofcollagen present in the ECM, may be adjusted by mixing the ECM producedby the cells of the invention with ECM of one or more other cell types.In addition, biologically active substances such as proteins, growthfactors and/or drugs, can be incorporated into the ECM. Exemplarybiologically active substances include tissue growth factors, such asTGF-beta, and the like, which promote healing and tissue repair at thesite of the injection. Such additional agents may be utilized in any ofthe embodiments described herein above, e.g., with whole cell lysates,soluble cell fractions, or further purified components and productsproduced by the PPDCs.

Pharmaceutical Compositions Comprising PPDCs, PPDC Components orProducts

In another aspect, the invention provides pharmaceutical compositionsthat utilize the PPDCs, PPDC populations, components and products ofPPDCs in various methods for treatment of neurodegenerative conditions.Certain embodiments encompass pharmaceutical compositions comprisinglive cells (PPDCs alone or admixed with other cell types). Otherembodiments encompass pharmaceutical compositions comprising PPDCcellular components (e.g., cell lysates, soluble cell fractions,conditioned medium, ECM, or components of any of the foregoing) orproducts (e.g., trophic and other biological factors produced naturallyby PPDCs or through genetic modification, conditioned medium from PPDCculture). In either case, the pharmaceutical composition may furthercomprise other active agents, such as anti-inflammatory agents,anti-apoptotic agents, antioxidants, growth factors, neurotrophicfactors or neuroregenerative or neuroprotective drugs as known in theart.

Examples of other components that may be added to PPDC pharmaceuticalcompositions include, but are not limited to: (1) other neuroprotectiveor neurobeneficial drugs; (2) selected extracellular matrix components,such as one or more types of collagen known in the art, and/or growthfactors, platelet-rich plasma, and drugs (alternatively, PPDCs may begenetically engineered to express and produce growth factors); (3)anti-apoptotic agents (e.g., erythropoietin (EPO), EPO mimetibody,thrombopoietin, insulin-like growth factor (IGF)-I, IGF-II, hepatocytegrowth factor, caspase inhibitors); (4) anti-inflammatory compounds(e.g., p38 MAP kinase inhibitors, TGF-beta inhibitors, statins, IL-6 andIL-1 inhibitors, PEMIROLAST, TRANILAST, REMICADE, SIROLIMUS, andnon-steroidal anti-inflammatory drugs (NSAIDS) (such as TEPOXALIN,TOLMETIN, and SUPROFEN); (5) immunosuppressive or immunomodulatoryagents, such as calcineurin inhibitors, mTOR inhibitors,antiproliferatives, corticosteroids and various antibodies; (6)antioxidants such as probucol, vitamins C and E, conenzyme Q-10,glutathione, L-cysteine and N-acetylcysteine; and (7) local anesthetics,to name a few.

Pharmaceutical compositions of the invention comprise PPDCs, orcomponents or products thereof, formulated with a pharmaceuticallyacceptable carrier or medium. Suitable pharmaceutically acceptablecarriers include water, salt solution (such as Ringer's solution),alcohols, oils, gelatins, and carbohydrates, such as lactose, amylose,or starch, fatty acid esters, hydroxymethylcellulose, and polyvinylpyrolidine. Such preparations can be sterilized, and if desired, mixedwith auxiliary agents such as lubricants, preservatives, stabilizers,wetting agents, emulsifiers, salts for influencing osmotic pressure,buffers, and coloring. Pharmaceutical carriers suitable for use in thepresent invention are known in the art and are described, for example,in Pharmaceutical Sciences (17^(th) Ed., Mack Pub. Co., Easton, Pa.) andWO 96/05309.

Typically, but not exclusively, pharmaceutical compositions comprisingPPDC components or products, but not live cells, are formulated asliquids (or as solid tablets, capsules and the like, when oral deliveryis appropriate). These may be formulated for administration by anyacceptable route known in the art to achieve delivery of drugs andbiological molecules to the target neural tissue, including, but notlimited to, oral, nasal, ophthalmic and parenteral, includingintravenous. Particular routes of parenteral administration include, butare not limited to, intramuscular, subcutaneous, intraperitoneal,intracerebral, intraventricular, intracerebroventricular, intrathecal,intracisternal, intraspinal and/or peri-spinal routes of administrationby delivery via intracranial or intravertebral needles and/or catheterswith or without pump devices.

Pharmaceutical compositions comprising PPDC live cells are typicallyformulated as liquids, semisolids (e.g., gels) or solids (e.g.,matrices, scaffolds and the like, as appropriate for neural tissueengineering). Liquid compositions are formulated for administration byany acceptable route known in the art to achieve delivery of live cellsto the target neural tissues. Typically, these include injection orinfusion into the CNS or PNS, either in a diffuse fashion or targeted tothe site of neurological disease or distress, by a route ofadministration including, but not limited to, intraocular,intracerebral, intraventricular, intracerebroventricular, intrathecal,intracisternal, intraspinal and/or peri-spinal routes of administrationby delivery via intracranial or intravertebral needles and/or catheterswith or without pump devices.

Pharmaceutical compositions comprising live cells in a semi-solid orsolid carrier are typically formulated for surgical implantation at thesite of neurological damage or distress. It will be appreciated thatliquid compositions also may be administered by surgical procedures. Inparticular embodiments, semi-solid or solid pharmaceutical compositionsmay comprise semi-permeable gels, lattices, cellular scaffolds and thelike, which may be non-biodegradable or biodegradable. For example, incertain embodiments, it may be desirable or appropriate to sequester theexogenous cells from their surroundings, yet enable the cells to secreteand deliver biological molecules (e.g. neurotrophic factors) tosurrounding neural cells. In these embodiments, cells may be formulatedas autonomous implants comprising living PPDCs or cell populationcomprising PPDCs surrounded by a non-degradable, selectively permeablebarrier that physically separates the transplanted cells from hosttissue. Such implants are sometimes referred to as “immunoprotective,”as they have the capacity to prevent immune cells and macromoleculesfrom killing the transplanted cells in the absence of pharmacologicallyinduced immunosuppression (for a review of such devices and methods,see, e.g., P. A. Tresco et al., 2000, Adv. Drug Delivery Rev. 42: 3-27).

In other embodiments, different varieties of degradable gels andnetworks are utilized for the pharmaceutical compositions of theinvention. For example, degradable materials particularly suitable forsustained release formulations include biocompatible polymers, such aspoly(lactic acid), poly (lactic-co-glycolic acid), methylcellulose,hyaluronic acid, collagen, and the like. The structure, selection anduse of degradable polymers in drug delivery vehicles have been reviewedin several publications, including, A. Domb et al., 1992, Polymers forAdvanced Technologies 3:279.

In other embodiments, e.g., for repair of large neural lesions, such asa damaged or severed spinal cord or a neural cord of a severed limb, itmay be desirable or appropriate to deliver the cells on or in abiodegradable, preferably bioresorbable or bioabsorbable, scaffold ormatrix. These typically three-dimensional biomaterials contain theliving cells attached to the scaffold, dispersed within the scaffold, orincorporated in an extracellular matrix entrapped in the scaffold. Onceimplanted into the target region of the body, these implants becomeintegrated with the host tissue, wherein the transplanted cellsgradually become established (see, e.g., P. A. Tresco et al., 2000,supra; see also D. W. Hutmacher, 2001, J. Biomater. Sci. Polymer Edn.12: 107-174).

Examples of scaffold or matrix (sometimes referred to collectively as“framework”) material that may be used in the present invention includenonwoven mats, porous foams, or self assembling peptides. Nonwoven matsmay, for example, be formed using fibers comprised of a syntheticabsorbable copolymer of glycolic and lactic acids (PGA/PLA), sold underthe tradename VICRYL (Ethicon, Inc., Somerville, N.J.), Foams, composedof, for example, poly(epsilon-caprolactone)/poly(glycolic acid)(PCL/PGA) copolymer, formed by processes such as freeze-drying, orlyophilized, as discussed in U.S. Pat. No. 6,355,699, also may beutilized. Hydrogels such as self-assembling peptides (e.g., RAD16) mayalso be used. In situ-forming degradable networks are also suitable foruse in the invention (see, e.g., Anseth, K. S. et al., 2002, J.Controlled Release 78: 199-209; Wang, D. et al., 2003, Biomaterials 24:3969-3980; U.S. Patent Publication 2002/0022676 to He et al.). Thesematerials are formulated as fluids suitable for injection, then may beinduced by a variety of means (e.g., change in temperature, pH, exposureto light) to form degradable hydrogel networks in situ or in vivo.

In another embodiment, the framework is a felt, which can be composed ofa multifilament yarn made from a bioabsorbable material, e.g., PGA, PLA,PCL copolymers or blends, or hyaluronic acid. The yarn is made into afelt using standard textile processing techniques consisting ofcrimping, cutting, carding and needling. In another embodiment, cellsare seeded onto foam scaffolds that may be composite structures.

In many of the abovementioned embodiments, the framework may be moldedinto a useful shape, such as that of the spinal cord with segregatedcolumns for nerve tract repair, for example (Friedman J A et al., 2002,Neurosurgery 51: 742-51). Furthermore, it will be appreciated that PPDCsmay be cultured on pre-formed, non-degradable surgical or implantabledevices, e.g., in a manner corresponding to that used for preparingfibroblast-containing GDC endovascular coils, for instance (Marx, W. F.et al., 2001, Am. J. Neuroradiol. 22: 323-333).

The matrix, scaffold or device may be treated prior to inoculation ofcells in order to enhance cell attachment. For example, prior toinoculation, nylon matrices can be treated with 0.1 molar acetic acidand incubated in polylysine, PBS, and/or collagen to coat the nylon.Polystyrene can be similarly treated using sulfuric acid. The externalsurfaces of a framework may also be modified to improve the attachmentor growth of cells and differentiation of tissue, such as by plasmacoating the framework or addition of one or more proteins (e.g.,collagens, elastic fibers, reticular fibers), glycoproteins,glycosaminoglycans (e.g., heparin sulfate, chondroitin-4-sulfate,chondroitin-6-sulfate, dermatan sulfate, keratin sulfate), a cellularmatrix, and/or other materials such as, but not limited to, gelatin,alginates, agar, agarose, and plant gums, among others.

PPDC-containing frameworks are prepared according to methods known inthe art. For example, cells can be grown freely in a culture vessel tosub-confluency or confluency, lifted from the culture and inoculatedonto the framework. Growth factors may be added to the culture mediumprior to, during, or subsequent to inoculation of the cells to triggerdifferentiation and tissue formation, if desired. Alternatively, theframeworks themselves may be modified so that the growth of cellsthereon is enhanced, or so that the risk of rejection of the implant isreduced. Thus, one or more biologically active compounds, including, butnot limited to, anti-inflammatories, immunosuppressants or growthfactors, may be added to the framework for local release.

Methods of using PPDCs, PPDC Components or Products

PPDCs, or cell populations comprising PPDCs, or components of orproducts produced by PPDCs, may be used in a variety of ways to supportand facilitate repair and regeneration of neural cells and tissues. Suchutilities encompass in vitro, ex vivo and in vivo methods.

In Vitro and Ex Vivo Methods:

In one embodiment, PPDCs may be used in vitro to screen a wide varietyof compounds for effectiveness and cytotoxicity of pharmaceuticalagents, growth factors, regulatory factors, and the like. For example,such screening may be performed on substantially homogeneous populationsof PPDCs to assess the efficacy or toxicity of candidate compounds to beformulated with, or co-administered with, the PPDCs, for treatment of aneurodegenerative condition. Alternatively, such screening may beperformed on PPDCs that have been stimulated to differentiate into aneural cell or neural progenitor cell, for the purpose of evaluating theefficacy of new pharmaceutical drug candidates. In this embodiment, thePPDCs are maintained in vitro and exposed to the compound to be tested.The activity of a potentially cytotoxic compound can be measured by itsability to damage or kill cells in culture. This may readily be assessedby vital staining techniques. The effect of growth or regulatory factorsmay be assessed by analyzing the number or robustness of the culturedcells, as compared with cells not exposed to the factors. This may beaccomplished using standard cytological and/or histological techniques,including the use of immunocytochemical techniques employing antibodiesthat define type-specific cellular antigens.

In a further embodiment, as discussed above, PPDCs can be cultured invitro to produce biological products that are either naturally producedby the cells, or produced by the cells when induced to differentiateinto neural lineages, or produced by the cells via genetic modification.For instance, TIMP1, TPO, KGF, HGF, FGF, HBEGF, BDNF, MIP1b, MCP1,RANTES, I309, TARC, MDC, and IL-8 were found to be secreted fromumbilicus-derived cells grown in Growth Medium. TIMP1, TPO, KGF, HGF,HBEGF, BDNF, MIP1a, MCP-1, RANTES, TARC, Eotaxin, and IL-8 were found tobe secreted from placenta-derived PPDCs cultured in Growth Medium (seeExamples). Some of these trophic factors, such as BDNF and IL-6, haveimportant roles in neural regeneration. Other trophic factors, as yetundetected or unexamined, of use in neural repair and regeneration, arelikely to be produced by PPDCs and possibly secreted into the medium.

In this regard, another embodiment of the invention features use ofPPDCs for production of conditioned medium, either from undifferentiatedPPDCs or from PPDCs incubated under conditions that stimulatedifferentiation into a neural lineage. Such conditioned media arecontemplated for use in in vitro or ex vivo culture of neurogeneicprecursor cells, or in vivo to support transplanted cells comprisinghomogeneous populations of PPDCs or heterogeneous populations comprisingPPDCs and neural progenitors, for example.

Yet another embodiment comprises the use of PPCD cell lysates, solublecell fractions or components thereof, or ECM or components thereof, fora variety of purposes. As mentioned above, some of these components maybe used in pharmaceutical compositions. In other embodiments, a celllysate or ECM is used to coat or otherwise treat substances or devicesto be used surgically, or for implantation, or for ex vivo purposes, topromote healing or survival of cells or tissues contacted in the courseof such treatments.

As described in Examples 13 and 15, PPDCs have demonstrated the abilityto support survival, growth and differentiation of adult neuralprogenitor cells when grown in co-culture with those cells. Accordingly,in another embodiment, PPDCs are used advantageously in co-cultures invitro to provide trophic support to other cells, in particular neuralcells and neural progenitors. For co-culture, it may be desirable forthe PPDCs and the desired other cells to be co-cultured under conditionsin which the two cell types are in contact. This can be achieved, forexample, by seeding the cells as a heterogeneous population of cells inculture medium or onto a suitable culture substrate. Alternatively, thePPDCs can first be grown to confluence, and then will serve as asubstrate for the second desired cell type in culture. In this latterembodiment, the cells may further be physically separated, e.g., by amembrane or similar device, such that the other cell type may be removedand used separately, following the co-culture period. Use of PPDCs inco-culture to promote expansion and differentiation of neural cell typesmay find applicability in research and in clinical/therapeutic areas.For instance, PPDC co-culture may be utilized to facilitate growth anddifferentiation of neural cells in culture, for basic research purposesor for use in drug screening assays, for example. PPDC co-culture mayalso be utilized for ex vivo expansion of neural progenitors for lateradministration for therapeutic purposes. For example, neural progenitorcells may be harvested from an individual, expanded ex vivo inco-culture with PPDCs, then returned to that individual (autologoustransfer) or another individual (syngeneic or allogeneic transfer). Inthese embodiments, it will be appreciated that, following ex vivoexpansion, the mixed population of cells comprising the PPDCs and neuralprogenitors could be administered to a patient in need of treatment.Alternatively, in situations where autologous transfer is appropriate ordesirable, the co-cultured cell populations may be physically separatedin culture, enabling removal of the autologous neural progenitors foradministration to the patient.

In Vivo Methods:

As set forth in Examples 16 and 17, PPDCs have been shown to beeffectively transplanted into the body, and to supply lost neuralfunction in an animal model accepted for its predictability of efficacyin humans. These results support a preferred embodiment of theinvention, wherein PPDCs are used in cell therapy for treating aneurodegenerative condition. Once transplanted into a target neurallocation in the body, PPDCs may themselves differentiate into one ormore neural phenotypes, or they may provide trophic support for neuralprogenitors and neural cells in situ, or they may exert a beneficialeffect in both of those fashions, among others.

PPDCs may be administered alone (e.g., as substantially homogeneouspopulations) or as admixtures with other cells. As described above,PPDCs may be administered as formulated in a pharmaceutical preparationwith a matrix or scaffold, or with conventional pharmaceuticallyacceptable carriers. Where PPDCs are administered with other cells, theymay be administered simultaneously or sequentially with the other cells(either before or after the other cells). Cells that may be administeredin conjunction with PPDCs include, but are not limited to, neurons,astrocytes, oligodendrocytes, neural progenitor cells, neural stem cellsand/or other multipotent or pluripotent stem cells. The cells ofdifferent types may be admixed with the PPDCs immediately or shortlyprior to administration, or they may be co-cultured together for aperiod of time prior to administration.

The PPDCs may be administered with other neuro-beneficial drugs orbiological molecules, or other active agents, such as anti-inflammatoryagents, anti-apoptotic agents, antioxidants, growth factors,neurotrophic factors or neuroregenerative or neuroprotective drugs asknown in the art. When PPDCs are administered with other agents, theymay be administered together in a single pharmaceutical composition, orin separate pharmaceutical compositions, simultaneously or sequentiallywith the other agents (either before or after administration of theother agents).

Examples of other components that may be administered with PPDCsinclude, but are not limited to: (1) other neuroprotective orneurobeneficial drugs; (2) selected extracellular matrix components,such as one or more types of collagen known in the art, and/or growthfactors, platelet-rich plasma, and drugs (alternatively, PPDCs may begenetically engineered to express and produce growth factors); (3)anti-apoptotic agents (e.g., erythropoietin (EPO), EPO mimetibody,thrombopoietin, insulin-like growth factor (IGF)-I, IGF-II, hepatocytegrowth factor, caspase inhibitors); (4) anti-inflammatory compounds(e.g., p38 MAP kinase inhibitors, TGF-beta inhibitors, statins, IL-6 andIL-1 inhibitors, PEMIROLAST, TRANILAST, REMICADE, SIROLIMUS, andnon-steroidal anti-inflammatory drugs (NSAIDS) (such as TEPOXALIN,TOLMETIN, and SUPROFEN); (5) immunosuppressive or immunomodulatoryagents, such as calcineurin inhibitors, mTOR inhibitors,antiproliferatives, corticosteroids and various antibodies; (6)antioxidants such as probucol, vitamins C and E, conenzyme Q-10,glutathione, L-cysteine and N-acetylcysteine; and (7) local anesthetics,to name a few.

In one embodiment, PPDCs are administered as undifferentiated cells,i.e., as cultured in Growth Medium. Alternatively, PPDCs may beadministered following exposure in culture to conditions that stimulatedifferentiation toward a desired neural phenotype, e.g., astrocyte,oligodendrocyte or neuron, and more specifically, serotoninergic,dopaminergic, cholinergic, GABA-ergic or glutamatergic neurons (see,e.g., Isacson, O., 2003. The Lancet (Neurology) 2: 417-424).

The cells of the invention may be surgically implanted, injected,delivered (e.g., by way of a catheter or syringe), or otherwiseadministered directly or indirectly to the site of neurological damageor distress. Routes of administration of the cells of the invention orcompositions thereof include, but are not limited to, intravenous,intramuscular, subcutaneous, intranasal, intracerebral,intraventricular, intracerebroventricular, intrathecal, intracisternal,intraspinal and/or peri-spinal routes of administration by delivery viaintracranial or intravertebral needles and/or catheters with or withoutpump devices.

When cells are administered in semi-solid or solid devices, surgicalimplantation into a precise location in the body is typically a suitablemeans of administration. Liquid or fluid pharmaceutical compositions,however, may be administered to a more general location in the CNS orPNS (e.g., throughout a diffusely affected area, such as would be thecase in Parkinson's disease or diffuse ischemic injury, for example),inasmuch as neural progenitor cells have been shown to be capable ofextensive migration from a point of entry to the nervous system to aparticular location, e.g., by following radial glia or by responding tochemical signals.

Indeed, this migratory ability of neural stem cells has opened a newavenue for treatment of malignant brain tumors, i.e., use of progenitorcells for delivery of therapeutic genes/gene products for the treatmentof these migratory tumors. For example, it has been reported that neuralstem cells, when implanted into intracranial gliomas in vivo in adultrodents, distribute themselves quickly and extensively through the tumorbed and migrate in juxtaposition to expanding and advancing tumor cells,while continuing to stably express a foreign gene (Aboody, K. et al.,2000, Proc. Natl. Acad. Sci. USA 97:12846-12851). PPDCs are alsoexpected to be suitable for this type of use, i.e., PPDCs geneticallymodified to produce an apoptotic or other antineoplastic agent, e.g.,IL-12 (Ehtesham, M. et al., 2002, Cancer Research 62: 5657-5663) ortumor necrosis factor-related apoptosis-inducing ligand (Ehtesham, M. etal., 2002, Cancer Research 62: 7170-7174) may be injected or otherwiseadministered to a general site of a malignant tumor (e.g.,glioblastoma), whereafter the PPDCs can migrate to the tumor cells forlocal delivery of the therapeutic agent.

Other embodiments encompass methods of treating neurodegenerativeconditions by administering pharmaceutical compositions comprising PPDCcellular components (e.g., cell lysates or components thereof) orproducts (e.g., trophic and other biological factors produced naturallyby PPDCs or through genetic modification, conditioned medium from PPDCculture). Again, these methods may further comprise administering otheractive agents, such as growth factors, neurotrophic factors orneuroregenerative or neuroprotective drugs as known in the art.

Dosage forms and regimes for administering PPDCs or any of the otherpharmaceutical compositions described herein are developed in accordancewith good medical practice, taking into account the condition of theindividual patient, e.g., nature and extent of the neurodegenerativecondition, age, sex, body weight and general medical condition, andother factors known to medical practitioners. Thus, the effective amountof a pharmaceutical composition to be administered to a patient isdetermined by these considerations as known in the art.

Because the CNS is a somewhat immunoprivileged tissue, it may not benecessary or desirable to immunosuppress a patient prior to initiationof cell therapy with PPDCs. In addition, as set forth in Example 11,PPDCs have been shown not to stimulate allogeneic PBMCs in a mixedlymphocyte reaction. Accordingly, transplantation with allogeneic, oreven xenogeneic, PPDCs may be tolerated in some instances.

However, in other instances it may be desirable or appropriate topharmacologically immunosuppress a patient prior to initiating celltherapy. This may be accomplished through the use of systemic or localimmunosuppressive agents, or it may be accomplished by delivering thecells in an encapsulated device, as described above. These and othermeans for reducing or eliminating an immune response to the transplantedcells are known in the art. As an alternative, PPDCs may be geneticallymodified to reduce their immunogenicity, as mentioned above.

Survival of transplanted PPDCs in a living patient can be determinedthrough the use of a variety of scanning techniques, e.g., computerizedaxial tomography (CAT or CT) scan, magnetic resonance imaging (MRI) orpositron emission tomography (PET) scans. Determination of transplantsurvival can also be done post mortem by removing the neural tissue, andexamining it visually or through a microscope. Alternatively, cells canbe treated with stains that are specific for neural cells or productsthereof, e.g., neurotransmitters. Transplanted cells can also beidentified by prior incorporation of tracer dyes such as rhodamine- orfluorescein-labeled microspheres, fast blue, ferric microparticles,bisbenzamide or genetically introduced reporter gene products, such asbeta-galactosidase or beta-glucuronidase.

Functional integration of transplanted PPDCs into neural tissue of asubject can be assessed by examining restoration of the neural functionthat was damaged or diseased. Such functions include, but are notlimited to motor, cognitive, sensory and endocrine functions, inaccordance with procedures well known to neurobiologists and physicians.

Kits and Banks Comprising PPDCs, PPDC Components or Products

In another aspect, the invention provides kits that utilize the PPDCs,PPDC populations, components and products of PPDCs in various methodsfor neural regeneration and repair as described above. Where used fortreatment of neurodegenerative conditions, or other scheduled treatment,the kits may include one or more cell populations, including at leastPPDCs and a pharmaceutically acceptable carrier (liquid, semi-solid orsolid). The kits also optionally may include a means of administeringthe cells, for example by injection. The kits further may includeinstructions for use of the cells. Kits prepared for field hospital use,such as for military use may include full-procedure supplies includingtissue scaffolds, surgical sutures, and the like, where the cells are tobe used in conjunction with repair of acute injuries. Kits for assaysand in vitro methods as described herein may contain one or more of (1)PPDCs or components or products of PPDCs, (2) reagents for practicingthe in vitro method, (3) other cells or cell populations, asappropriate, and (4) instructions for conducting the in vitro method.

In yet another aspect, the invention also provides for banking oftissues, cells, cellular components and cell populations of theinvention. As discussed above, the cells are readily cryopreserved. Theinvention therefore provides methods of cryopreserving the cells in abank, wherein the cells are stored frozen and associated with a completecharacterization of the cells based on immunological, biochemical andgenetic properties of the cells. The frozen cells can be thawed andexpanded or used directly for autologous, syngeneic, or allogeneictherapy, depending on the requirements of the procedure and the needs ofthe patient. Preferably, the information on each cryopreserved sample isstored in a computer, which is searchable based on the requirements ofthe surgeon, procedure and patient with suitable matches being madebased on the characterization of the cells or populations. Preferably,the cells of the invention are grown and expanded to the desiredquantity of cells and therapeutic cell compositions are prepared eitherseparately or as co-cultures, in the presence or absence of a matrix orsupport. While for some applications it may be preferable to use cellsfreshly prepared, the remainder can be cryopreserved and banked byfreezing the cells and entering the information in the computer toassociate the computer entry with the samples. Even where it is notnecessary to match a source or donor with a recipient of such cells, forimmunological purposes, the bank system makes it easy to match, forexample, desirable biochemical or genetic properties of the banked cellsto the therapeutic needs. Upon matching of the desired properties with abanked sample, the sample is retrieved and prepared for therapeutic use.Cell lysates, ECM or cellular components prepared as described hereinmay also be cryopreserved or otherwise preserved (e.g., bylyophilization) and banked in accordance with the present invention.

The following examples are provided to describe the invention in greaterdetail. They are intended to illustrate, not to limit, the invention.

As used in the following examples and elsewhere in the specification,the term Growth Medium generally refers to a medium sufficient for theculturing of PPDCs. In particular, one presently preferred medium forthe culturing of the cells of the invention in comprises Dulbecco'sModified Essential Media (also abbreviated DMEM herein). Particularlypreferred is DMEM-low glucose (also DMEM-LG herein) (Invitrogen,Carlsbad, Calif.). The DMEM-low glucose is preferably supplemented with15% (v/v) fetal bovine serum (e.g. defined fetal bovine serum, Hyclone,Logan Utah), antibiotics/antimycotics ((preferably 50-100Units/milliliter penicillin, 50-100 microgram/milliliter streptomycin,and 0-0.25 microgram/milliliter amphotericin B; Invitrogen, Carlsbad,Calif.)), and 0.001% (v/v) 2-mercaptoethanol (Sigma, St. Louis Mo.). Asused in the Examples below, Growth Medium refers to DMEM-low glucosewith 15% fetal bovine serum and antibiotics/antimycotics (whenpenicillin/streptomycin are included, it is preferably at 50U/milliliter and 50 microgram/milliliter respectively; whenpenicillin/streptomycin/amphotericin B are use, it is preferably at 100U/milliliter, 100 microgram/milliliter and 0.25 microgram/milliliter,respectively). In some cases different growth media are used, ordifferent supplementations are provided, and these are normallyindicated in the text as supplementations to Growth Medium.

Also relating to the following examples and used elsewhere in thespecification, the term standard growth conditions refers to culturingof cells at 37° C., in a standard atmosphere comprising 5% CO₂. Whileforegoing the conditions are useful for culturing, it is to beunderstood that such conditions are capable of being varied by theskilled artisan who will appreciate the options available in the art forculturing cells.

The following abbreviations may appear in the examples and elsewhere inthe specification and claims: ANG2 (or Ang2) for angiopoietin 2; APC forantigen-presenting cells; BDNF for brain-derived neurotrophic factor;bFGF for basic fibroblast growth factor; bid (BID) for “bis in die”(twice per day); CK18 for cytokeratin 18; CNS for central nervoussystem; CXC ligand 3 for chemokine receptor ligand 3; DMEM forDulbecco's Minimal Essential Medium; DMEM:lg (or DMEM:Lg, DMEM:LG) forDMEM with low glucose; EDTA for ethylene diamine tetraacetic acid; EGF(or E) for epidermal growth factor; FACS for fluorescent activated cellsorting; FBS for fetal bovine serum; FGF (or F) for fibroblast growthfactor; GCP-2 for granulocyte chemotactic protein-2; GFAP for glialfibrillary acidic protein; HB-EGF for heparin-binding epidermal growthfactor; HCAEC for Human coronary artery endothelial cells; HGF forhepatocyte growth factor; hMSC for Human mesenchymal stem cells;HNF-1alpha for hepatocyte-specific transcription factor 1 alpha; HUVECfor Human umbilical vein endothelial cells; I309 for a chemokine and theligand for the CCR8 receptor; IGF-1 for insulin-like growth factor 1;IL-6 for interleukin-6; IL-8 for interleukin 8; K19 for keratin 19; K8for keratin 8; KGF for keratinocyte growth factor; LIF for leukemiainhibitory factor; MBP for myelin basic protein; MCP-1 for monocytechemotactic protein 1; MDC for macrophage-derived chemokine; MIPlalphafor macrophage inflammatory protein 1 alpha; MIP1beta for macrophageinflammatory protein 1 beta; MMP for matrix metalloprotease (MMP); MSCfor mesenchymal stem cells; NHDF for Normal Human Dermal Fibroblasts;NPE for Neural Progenitor Expansion media; O4 for oligodendrocyte orglial differentiation marker O4; PBMC for Peripheral blood mononuclearcell; PBS for phosphate buffered saline; PDGFbb for platelet derivedgrowth factor; PO for “per os” (by mouth); PNS for peripheral nervoussystem; Rantes (or RANTES) for regulated on activation, normal T cellexpressed and secreted; rhGDF-5 for recombinant human growth anddifferentiation factor 5; SC for subcutaneously; SDF-1alpha forstromal-derived factor 1 alpha; SHH for sonic hedgehog; SOP for standardoperating procedure; TARC for thymus and activation-regulated chemokine;TCP for Tissue culture plastic; TCPS for tissue culture polystyrene;TGFbeta2 for transforming growth factor beta2; TGF beta-3 fortransforming growth factor beta-3; TIMP1 for tissue inhibitor of matrixmetalloproteinase 1; TPO for thrombopoietin; TuJ1 for BIII Tubulin; VEGFfor vascular endothelial growth factor; vWF for von Willebrand factor;and alphaFP for alpha-fetoprotein.

EXAMPLE 1 Derivation of Cells from Postpartum Tissue

This example describes the preparation of postpartum-derived cells fromplacental and umbilical cord tissues. Postpartum umbilical cords andplacentae were obtained upon birth of either a full term or pre-termpregnancy. Cells were harvested from 5 separate donors of umbilicus andplacental tissue. Different methods of cell isolation were tested fortheir ability to yield cells with: 1) the potential to differentiateinto cells with different phenotypes, a characteristic common to stemcells, or 2) the potential to provide trophic factors useful for othercells and tissues.

Methods & Materials

Umbilical cell isolation. Umbilical cords were obtained from NationalDisease Research Interchange (NDRI, Philadelphia, Pa.). The tissues wereobtained following normal deliveries. The cell isolation protocol wasperformed aseptically in a laminar flow hood. To remove blood anddebris, the cord was washed in phosphate buffered saline (PBS;Invitrogen, Carlsbad, Calif.) in the presence of antimycotic andantibiotic (100 units/milliliter penicillin, 100 micrograms/milliliterstreptomycin, 0.25 micrograms/milliliter amphotericin B). The tissueswere then mechanically dissociated in 150 cm² tissue culture plates inthe presence of 50 milliliters of medium (DMEM-Low glucose or DMEM-Highglucose; Invitrogen), until the tissue was minced into a fine pulp. Thechopped tissues were transferred to 50 milliliter conical tubes(approximately 5 grams of tissue per tube). The tissue was then digestedin either DMEM-Low glucose medium or DMEM-High glucose medium, eachcontaining antimycotic and antibiotic as described above. In someexperiments, an enzyme mixture of collagenase and dispase was used(“C:D;” collagenase (Sigma, St Louis, Mo.), 500 Units/milliliter; anddispase (Invitrogen), 50 Units/milliliter in DMEM:-Low glucose medium).In other experiments a mixture of collagenase, dispase and hyaluronidase(“C:D:H”) was used (collagenase, 500 Units/milliliter; dispase, 50Units/milliliter; and hyaluronidase (Sigma), 5 Units/milliliter, inDMEM:-Low glucose). The conical tubes containing the tissue, medium anddigestion enzymes were incubated at 37° C. in an orbital shaker(Environ, Brooklyn, N.Y.) at 225 rpm for 2 hrs.

After digestion, the tissues were centrifuged at 150×g for 5 minutes,the supernatant was aspirated. The pellet was resuspended in 20milliliters of Growth Medium (DMEM:Low glucose (Invitrogen), 15 percent(v/v) fetal bovine serum (FBS; defined bovine serum; Lot#AND18475;Hyclone, Logan, Utah), 0.001% (v/v) 2-mercaptoethanol (Sigma), 1milliliter per 100 milliliters of antibiotic/antimycotic as describedabove. The cell suspension was filtered through a 70-micrometer nyloncell strainer (BD Biosciences). An additional 5 milliliters rinsecomprising Growth Medium was passed through the strainer. The cellsuspension was then passed through a 40-micrometer nylon cell strainer(BD Biosciences) and chased with a rinse of an additional 5 millilitersof Growth Medium.

The filtrate was resuspended in Growth Medium (total volume 50milliliters) and centrifuged at 150×g for 5 minutes. The supernatant wasaspirated and the cells were resuspended in 50 milliliters of freshGrowth Medium. This process was repeated twice more.

Upon the final centrifugation supernatant was aspirated and the cellpellet was resuspended in 5 milliliters of fresh Growth Medium. Thenumber of viable cells was determined using Trypan Blue staining. Cellswere then cultured under standard conditions.

The cells isolated from umbilical cords were seeded at 5,000 cells/cm²onto gelatin-coated T-75 cm² flasks (Corning Inc., Corning, N.Y.) inGrowth Medium with antibiotics/antimycotics as described above. After 2days (in various experiments, cells were incubated from 2-4 days), spentmedium was aspirated from the flasks. Cells were washed with PBS threetimes to remove debris and blood-derived cells. Cells were thenreplenished with Growth Medium and allowed to grow to confluence (about10 days from passage 0) to passage 1. On subsequent passages (frompassage 1 to 2 and so on), cells reached sub-confluence (75-85 percentconfluence) in 4-5 days. For these subsequent passages, cells wereseeded at 5000 cells/cm². Cells were grown in a humidified incubatorwith 5 percent carbon dioxide and atmospheric oxygen, at 37° C.

Placental Cell Isolation. Placental tissue was obtained from NDRI(Philadelphia, Pa.). The tissues were from a pregnancy and were obtainedat the time of a normal surgical delivery. Placental cells were isolatedas described for umbilical cell isolation.

The following example applies to the isolation of separate populationsof maternal-derived and neonatal-derived cells from placental tissue.

The cell isolation protocol was performed aseptically in a laminar flowhood. The placental tissue was washed in phosphate buffered saline (PBS;Invitrogen, Carlsbad, Calif.) in the presence of antimycotic andantibiotic (as described above) to remove blood and debris. Theplacental tissue was then dissected into three sections: top-line(neonatal side or aspect), mid-line (mixed cell isolation neonatal andmaternal) and bottom line (maternal side or aspect).

The separated sections were individually washed several times in PBSwith antibiotic/antimycotic to further remove blood and debris. Eachsection was then mechanically dissociated in 150 cm² tissue cultureplates in the presence of 50 milliliters of DMEM/Low glucose, to a finepulp. The pulp was transferred to 50 milliliter conical tubes. Each tubecontained approximately 5 grams of tissue. The tissue was digested ineither DMEM-Low glucose or DMEM-High glucose medium containingantimycotic and antibiotic (100 U/milliliter penicillin, 100micrograms/milliliter streptomycin, 0.25 micrograms/milliliteramphotericin B) and digestion enzymes. In some experiments an enzymemixture of collagenase and dispase (“C:D”) was used containingcollagenase (Sigma, St Louis, Mo.) at 500 Units/milliliter and dispase(Invitrogen) at 50 Units/milliliter in DMEM-Low glucose medium. In otherexperiments a mixture of collagenase, dispase and hyaluronidase (C:D:H)was used (collagenase, 500 Units/milliliter; dispase, 50Units/milliliter; and hyaluronidase (Sigma), 5 Units/milliliter inDMEM-Low glucose). The conical tubes containing the tissue, medium, anddigestion enzymes were incubated for 2 h at 37° C. in an orbital shaker(Environ, Brooklyn, N.Y.) at 225 rpm.

After digestion, the tissues were centrifuged at 150×g for 5 minutes,the resultant supernatant was aspirated off. The pellet was resuspendedin 20 milliliters of Growth Medium withpenicillin/streptomycin/amphotericin B. The cell suspension was filteredthrough a 70 micometer nylon cell strainer (BD Biosciences), chased by arinse with an additional 5 milliliters of Growth Medium. The total cellsuspension was passed through a 40 micometer nylon cell strainer (BDBiosciences) followed with an additional 5 milliliters of Growth Mediumas a rinse.

The filtrate was resuspended in Growth Medium (total volume 50milliliters) and centrifuged at 150×g for 5 minutes. The supernatant wasaspirated and the cell pellet was resuspended in 50 milliliters of freshGrowth Medium. This process was repeated twice more. After the finalcentrifugation, supernatant was aspirated and the cell pellet wasresuspended in 5 milliliters of fresh Growth Medium. A cell count wasdetermined using the Trypan Blue Exclusion test. Cells were thencultured at standard conditions.

LIBERASE Cell Isolation. Cells were isolated from umbilicus tissues inDMEM-Low glucose medium with LIBERASE (Boehringer Mannheim Corp.,Indianapolis, Ind.) (2.5 milligrams per milliliter, Blendzyme 3; RocheApplied Sciences, Indianapolis, Ind.) and hyaluronidase (5Units/milliliter, Sigma). Digestion of the tissue and isolation of thecells was as described for other protease digestions above, using theLIBERASE/hyaluronidase mixture in place of the C:D or C:D:H enzymemixture. Tissue digestion with LIBERASE resulted in the isolation ofcell populations from postpartum tissues that expanded readily.

Cell isolation using other enzyme combinations. Procedures were comparedfor isolating cells from the umbilical cord using differing enzymecombinations. Enzymes compared for digestion included: i) collagenase;ii) dispase; iii) hyaluronidase; iv) collagenase:dispase mixture (C;D);v) collagenase:hyaluronidase mixture (C:H); vi) dispase:hyaluronidasemixture (D:H); and vii) collagenase:dispase:hyaluronidase mixture(C:D:H). Differences in cell isolation utilizing these different enzymedigestion conditions were observed (Table 1-1).

TABLE 1-1 Isolation of cells from umbilical cord tissue using varyingenzyme combinations Cells Cell Enzyme Digest Isolated ExpansionCollagenase X X Dispase + (>10 h) + Hyaluronidase X XCollagenase:Dispase ++ (<3 h) ++ Collagenase:Hyaluronidase ++ (<3 h) +Dispase:Hyaluronidase + (>10 h) + Collagenase:Dispase:Hyaluronidase +++(<3 h) +++ Key: + = good, ++ = very good, +++ = excellent, X = nosuccess under conditions tested

Isolation of cells from residual blood in the cords. Other attempts weremade to isolate pools of cells from umbilical cord by differentapproaches. In one instance umbilical cord was sliced and washed withGrowth Medium to dislodge the blood clots and gelatinous material. Themixture of blood, gelatinous material and Growth Medium was collectedand centrifuged at 150×g. The pellet was resuspended and seeded ontogelatin-coated flasks in Growth Medium. From these experiments a cellpopulation was isolated that readily expanded.

Isolation of cells from cord blood. Cells have also been isolated fromcord blood samples attained from NDRI. The isolation protocol used herewas that of International Patent Application PCT/US2002/029971 by Ho etal (Ho, T. W. et al., WO2003025149 A2). Samples (50 milliliter and 10.5milliliters, respectively) of umbilical cord blood (NDR1, PhiladelphiaPa.) were mixed with lysis buffer (filter-sterilized 155 mM ammoniumchloride, 10 millimolar potassium bicarbonate, 0.1 millimolar EDTAbuffered to pH 7.2 (all components from Sigma, St. Louis, Mo.)). Cellswere lysed at a ratio of 1:20 cord blood to lysis buffer. The resultingcell suspension was vortexed for 5 seconds, and incubated for 2 minutesat ambient temperature. The lysate was centrifuged (10 minutes at200×g). The cell pellet was resuspended in complete minimal essentialmedium (Gibco, Carlsbad Calif.) containing 10 percent fetal bovine serum(Hyclone, Logan Utah), 4 millimolar glutamine (Mediatech Herndon, Va.),100 Units penicillin per 100 milliliters and 100 micrograms streptomycinper 100 milliliters (Gibco, Carlsbad, Calif.). The resuspended cellswere centrifuged (10 minutes at 200×g), the supernatant was aspirated,and the cell pellet was washed in complete medium. Cells were seededdirectly into either T75 flasks (Corning, N.Y.), T75 laminin-coatedflasks, or T175 fibronectin-coated flasks (both Becton Dickinson,Bedford, Mass.).

Isolation of cells using different enzyme combinations and growthconditions. To determine whether cell populations could be isolatedunder different conditions and expanded under a variety of conditionsimmediately after isolation, cells were digested in Growth Medium withor without 0.001 percent (v/v) 2-mercaptoethanol (Sigma, St. Louis,Mo.), using the enzyme combination of C:D:H, according to the proceduresprovided above. Placental-derived cells so isolated were seeded under avariety of conditions. All cells were grown in the presence ofpenicillin/streptomycin. (Table 1-2).

TABLE 1-2 Isolation and culture expansion of postpartum cells undervarying conditions: Growth Condition Medium 15% FBS BME Gelatin 20% O2Factors 1 DMEM-Lg Y Y Y Y N 2 DMEM-Lg Y Y Y N (5%) N 3 DMEM-Lg Y Y N Y N4 DMEM-Lg Y Y N N (5%) N 5 DMEM-Lg N (2%) Y N (Laminin) Y EGF/FGF (20ng/mL) 6 DMEM-Lg N (2%) Y N (Laminin) N (5%) EGF/FGF (20 ng/mL) 7DMEM-Lg N (2%) Y N (Fibrone) Y PDGF/VEGF 8 DMEM-Lg N (2%) Y N (Fibrone)N (5%) PDGF/VEGF 9 DMEM-Lg Y N Y Y N 10 DMEM-Lg Y N Y N (5%) N 11DMEM-Lg Y N N Y N 12 DMEM-Lg Y N N N (5%) N 13 DMEM-Lg N (2%) N N(Laminin) Y EGF/FGF (20 ng/mL) 14 DMEM-Lg N (2%) N N (Laminin) N (5%)EGF/FGF (20 ng/mL) 15 DMEM-Lg N (2%) N N (Fibrone) Y PDGF/VEGF 16DMEM-Lg N (2%) N N (Fibrone) N (5%) PDGF/VEGF

Isolation of cells using different enzyme combinations and growthconditions. In all conditions cells attached and expanded well betweenpassage 0 and 1 (Table 1-2). Cells in conditions 5-8 and 13-16 weredemonstrated to proliferate well up to 4 passages after seeding at whichpoint they were cryopreserved and banked.

Cell isolation using different enzyme combinations. The combination ofC:D:H, provided the best cell yield following isolation, and generatedcells which expanded for many more generations in culture than the otherconditions (Table 1). An expandable cell population was not attainedusing collagenase or hyaluronidase alone. No attempt was made todetermine if this result is specific to the collagen that was tested.

Isolation of cells using different enzyme combinations and growthconditions. Cells attached and expanded well between passage 0 and 1under all conditions tested for enzyme digestion and growth (Table 2).Cells in experimental conditions 5-8 and 13-16 proliferated well up to 4passages after seeding, at which point they were cryopreserved. Allcells were banked for further investigation.

Isolation of cells from residual blood in the cords. Nucleated cellsattached and grew rapidly. These cells were analyzed by flow cytometryand were similar to cells obtained by enzyme digestion.

Isolation of cells from cord blood. The preparations contained red bloodcells and platelets. No nucleated cells attached and divided during thefirst 3 weeks. The medium was changed 3 weeks after seeding and no cellswere observed to attach and grow.

Summary. Populations of cells can be derived from umbilical cord andplacental tissue efficiently using the enzyme combination collagenase (amatrix metalloprotease), dispase (a neutral protease) and hyaluronidase(a mucolytic enzyme that breaks down hyaluronic acid). LIBERASE, whichis a Blendzyme, may also be used. Specifically, Blendzyme 3, which iscollagenase (4 Wunsch units/g) and thermolysin (1714 casein Units/g) wasalso used together with hyaluronidase to isolate cells. These cellsexpanded readily over many passages when cultured in Growth Medium ongelatin coated plastic.

Cells were also isolated from residual blood in the cords, but not cordblood. The presence of cells in blood clots washed from the tissue, thatadhere and grow under the conditions used, may be due to cells beingreleased during the dissection process.

EXAMPLE 2 Growth Characteristics of Postpartum-Derived Cells

The cell expansion potential of postpartum-derived cells (PPDCs) wascompared to other populations of isolated stem cells. The process ofcell expansion to senescence is referred to as Hayflick's limit(Hayflick L. 1974a, 1974b). Postpartum-derived cells are highly suitedfor therapeutic use because they can be readily expanded to sufficientcell numbers.

Materials and Methods

Gelatin-coating flasks. Tissue culture plastic flasks were coated byadding 20 milliliters 2% (w/v) porcine gelatin (Type B: 225 Bloom;Sigma, St Louis, Mo.) to a T75 flask (Corning, Corning, N.Y.) for 20minutes at room temperature. After removing the gelatin solution, 10milliliters phosphate-buffered saline (PBS) (Invitrogen, Carlsbad,Calif.) was added and then aspirated.

Comparison of expansion potential of PPDCs with other cell populations.For comparison of growth expansion potential the following cellpopulations were utilized; i) Mesenchymal stem cells (MSC; Cambrex,Walkersville, Md.); ii) Adipose-derived cells (U.S. Pat. No. 6,555,374B1; U.S. Patent Application US20040058412); iii) Normal dermal skinfibroblasts (cc-2509 lot # 9F0844; Cambrex, Walkersville, Md.); iv)Umbilicus-derived cells; and v) Placenta-derived cells. Cells wereinitially seeded at 5,000 cells/cm² on gelatin-coated T75 flasks inGrowth Medium with penicillin/streptomycin/amphotericin B. Forsubsequent passages, cell cultures were treated as follows. Aftertrypsinization, viable cells were counted after Trypan Blue staining.Cell suspension (50 microliters) was combined with Trypan Blue (50milliliters, Sigma, St. Louis Mo.). Viable cell numbers were estimatedusing a hemocytometer.

Following counting, cells were seeded at 5,000 cells/cm² ontogelatin-coated T 75 flasks in 25 milliliters of fresh Growth Medium.Cells were grown under standard conditions at 37° C. The Growth Mediumwas changed twice per week. When cells reached about 85 percentconfluence they were passaged; this process was repeated until the cellsreached senescence.

At each passage, cells were trypsinized and counted. The viable cellyield, population doubling [In (cell final/cell initial)/ln 2] anddoubling time (time in culture (h)/population doubling) were calculated.For the purposes of determining optimal cell expansion, the total cellyield per passage was determined by multiplying the total yield for theprevious passage by the expansion factor for each passage (i.e.,expansion factor=cell final/cell initial).

Expansion potential of cell banks at low density. The expansionpotential of cells banked at passage 10 was also tested, using adifferent set of conditions. Normal dermal skin fibroblasts (cc-2509 lot# 9F0844; Cambrex, Walkersville, Md.), umbilicus-derived cells, andplacenta-derived cells were tested. These cell populations had beenbanked at passage 10 previously, having been cultured at 5,000 cells/cm²and grown to confluence at each passage to that point. The effect ofcell density on the cell populations following cell thaw at passage 10was determined. Cells were thawed under standard conditions and countedusing Trypan Blue staining. Thawed cells were then seeded at 1000cells/cm² in DMEM:Low glucose Growth Medium with antibiotic/antimycoticas described above. Cells were grown under standard atmosphericconditions at 37° C. Growth Medium was changed twice a week and cellswere passaged as they reached about 85% confluence. Cells weresubsequently passaged until senescence, i.e., until they could not beexpanded any further. Cells were trypsinized and counted at eachpassage. The cell yield, population doubling (In (cell final/cellinitial)/ln 2) and doubling time (time in culture (h)/populationdoubling). The total cell yield per passage was determined bymultiplying total yield for the previous passage by the expansion factorfor each passage (i.e., expansion factor=cell final/cell initial).

Expansion of PPDCs at low density from initial cell seeding. Theexpansion potential of freshly isolated PPDCs under low cell seedingconditions was tested. PPDDs were prepared as described herein. Cellswere seeded at 1000 cells/cm² and passaged as described above untilsenescence. Cells were grown under standard atmospheric conditions at37° C. Growth Medium was changed twice per week. Cells were passaged asthey reached about 85% confluence. At each passage, cells weretrypsinized and counted by Trypan Blue staining. The cell yield,population doubling (In (cell final/cell initial)/ln 2) and doublingtime (time in culture (h)/population doubling) were calculated for eachpassage. The total cell yield per passage was determined by multiplyingthe total yield for the previous passage by the expansion factor foreach passage (i.e. expansion factor=cell final/cell initial). Cells weregrown on gelatin and non-gelatin coated flasks.

Expansion of clonal neonatal placenta-derived cells. Cloning was used inorder to expand a population of neonatal cells from placental tissue.Following isolation of three differential cell populations from theplacenta (as described herein), these cell populations were expandedunder standard growth conditions and then karyotyped to reveal theidentity of the isolated cell populations. Because the cells wereisolated from a mother who delivered a boy, it was straightforward todistinguish between the male and female chromosomes by performingmetaphase spreads. These experiments demonstrated that fetal-aspectcells were karyotype positive for neonatal phenotpye, mid-layer cellswere karyotype positive for both neonatal and maternal phenotypes andmaternal-aspect cells were karyotype positive for maternal cells.

Expansion of cells in low oxygen culture conditions. It has beendemonstrated that low oxygen cell culture conditions can improve cellexpansion in certain circumstances (US20040005704). To determine if cellexpansion of PPDCs could be improved by altering cell cultureconditions, cultures of umbilical-derived cells were grown in low oxygenconditions. Cells were seeded at 5000 cells/cm² in Growth Medium ongelatin coated flasks. Cells were initially cultured under standardatmospheric conditions through passage 5, at which point they weretransferred to low oxygen (5% O₂) culture conditions.

Other growth conditions. In other protocols, cells were expanded onnon-coated, collagen-coated, fibronectin-coated, laminin-coated andextracellular matrix protein-coated plates. Cultures have beendemonstrated to expand well on these different matrices.

Results

Comparison of expansion potential of PPDCs with other stem cell andnon-stem cell populations. Both umbilical-derived and placenta-derivedcells expanded for greater than 40 passages generating cell yieldsof >1E17 cells in 60 days. In contrast, MSCs and fibroblasts senescedafter <25 days and <60 days, respectively. Although adipose-derivedcells expanded for almost 60 days they generated total cell yields of4.5E12. Thus, when seeded at 5000 cells/cm² under the experimentalconditions utilized, postpartum-derived cells expanded much better thanthe other cell types grown under the same conditions (Table 2-1).

TABLE 2-1 Growth characteristics for different cell populations grown tosenescence Total Population Total Cell Cell Type Senescence DoublingsYield MSC 24 d 8 4.72 E7 Adipose 57 d 24 4.5 E12 Fibroblasts 53 d 262.82 E13 Umbilicus 65 d 42 6.15 E17 Placenta 80 d 46 2.49 E19

Expansion potential of cell banks at low density. Umbilicus-derived,placenta-derived and fibroblast cells expanded for greater than 10passages generating cell yields of >1E11 cells in 60 days (Table 2-2).After 60 days under these conditions the fibroblasts became senescentwhereas the umbilicus-derived and placenta-derived cell populationssenesced after 80 days, completing >50 and >40 population doublingsrespectively.

TABLE 2-2 Growth characteristics for different cell populations usinglow density growth expansion from passage 10 till senescence TotalPopulation Total Cell Cell Type Senescence Doublings Yield Fibroblast(P10) 80 d 43.68 2.59 E11 Umbilicus (P10) 80 d 53.6 1.25 E14 Placenta(P10) 60 d 32.96 6.09 E12

Expansion of PPDCs at low density from initial cell seeding. PPDCs wereexpanded at low density (1,000 cells/cm²) on gelatin-coated and uncoatedplates or flasks. Growth potential of these cells under these conditionswas good. The cells expanded readily in a log phase growth. The rate ofcell expansion was similar to that observed when placenta-derived cellswere seeded at 5000 cells/cm² on gelatin-coated flasks in Growth Medium.No differences were observed in cell expansion potential betweenculturing on either uncoated flasks or gelatin-coated flasks. However,cells appeared phenotypically much smaller on gelatin-coated flasks andmore larger cell phenotypes were observed on uncoated flasks.

Expansion of clonal neonatal or maternal placenta-derived cells. Aclonal neonatal or maternal cell population can be expanded fromplacenta-derived cells isolated from the neonatal aspect or the maternalaspect, respectively, of the placenta. Cells are serially diluted andthen seeded onto gelatin-coated plates in Growth medium for expansion at1 cell/well in 96-well gelatin coated plates. From this initial cloning,expansive clones are identified, trypsinized, and reseeded in 12-wellgelatin-coated plates in Growth medium and then subsequently passagedinto T25 gelatin-coated flasks at 5,000 cells/cm² in Growth medium.Subcloning is performed to ensure that a clonal population of cells hasbeen identified. For subcloning experiments, cells are trypsinized andreseeded at 0.5 cells/well. The subclones that grow well are expanded ingelatin-coated T25 flasks at 5,000 cells cm²/flask. Cells are passagedat 5,000 cells cm²/T75 flask. The growth characteristics of a clone maybe plotted to demonstrate cell expansion. Karyotyping analysis canconfirm that the clone is either neonatal or maternal.

Expansion of cells in low oxygen culture conditions. Cells expanded wellunder the reduced oxygen conditions, however, culturing under low oxygenconditions did not appear to have a significant effect on cell expansionof PPDCs under the conditions used.

Summary. Cell expansion conditions comprising growing isolatedpostpartum-derived cells at densities of about 5000 cells/cm², in GrowthMedium on gelatin-coated or uncoated flasks, under standard atmosphericoxygen, are sufficient to generate large numbers of cells at passage 11.Furthermore, the data suggests that the cells can be readily expandedusing lower density culture conditions (e.g. 1000 cells/cm²).Postpartum-derived cell expansion in low oxygen conditions alsofacilitates cell expansion, although no incremental improvement in cellexpansion potential has yet been observed when utilizing theseconditions for growth. Presently, culturing postpartum-derived cellsunder standard atmospheric conditions is preferred for generating largepools of cells. However, when the culture conditions are altered,postpartum-derived cell expansion can likewise be altered. This strategymay be used to enhance the proliferative and differentiative capacity ofthese cell populations.

Under the conditions utilized, while the expansion potential of MSC andadipose-derived cells is limited, postpartum-derived cells expandreadily to large numbers.

References for Example 2

1) Hayflick L. 1974a. J Am Geriatr Soc. 22:1-12.

2) Hayflick L. 1974b. Gerontologist. 14:37-45.

3) U.S. Patent publication US20040058412

4) U.S. Patent publication US20040048372

5) U.S. Patent publication US20040005704.

EXAMPLE 3 Evaluation of Growth Media for Placenta-Derived Cells

Several cell culture media were evaluated for their ability to supportthe growth of placenta-derived cells. The growth of placenta-derivedcells in normal (20%) and low (5%) oxygen was assessed after 3 daysusing the MTS calorimetric assay.

Methods & Materials

Placenta-derived cells at passage 8 (P8) were seeded at 1×10³ cells/wellin 96 well plates in Growth Medium with penicillin/streptomycin. After 8hours the medium was changed as described below and cells were incubatedin normal (atmospheric) or low (5%, v/v) oxygen at 37° C., 5% CO₂ for 48hours. MTS was added to the culture medium (CELLTITER 96 AQueous OneSolution Cell Proliferation Assay, Promega, Madison, Wis.) for 3 hoursand the absorbance measured at 490 nanometers (Molecular Devices,Sunnyvale Calif.).

TABLE 3-1 Culture Media Added fetal bovine Culture Medium Supplier serum% (v/v) DMEM low glucose Gibco Carlsbad CA 0, 2 10 DMEM high glucoseGibco 0, 2 10 RPMI 1640 Mediatech, Inc. 0, 2 10 Herndon, VA Cellgro-free (Serum-free, Mediatech, Inc. — Protein-free Ham's F10Mediatech, Inc. 0, 2 10 MSCGM (complete with Cambrex, 0, 2 10 serum)Walkersville, MD Complete-serum free Mediatech, Inc. — w/albumin GrowthMedium NA — Ham's F12 Mediatech, Inc. 0, 2 10 Iscove's Mediatech, Inc.0, 2 10 Basal Medium Eagle's Mediatech, Inc. DMEM/F12 (1:1) Mediatech,Inc. 0, 2 10Results

Standard curves for the MTS assay established a linear correlationbetween an increase in absorbance and an increase in cell number. Theabsorbance values obtained were converted into estimated cell numbersand the change (%) relative to the initial seeding was calculated.

The Effect of Serum. The addition of serum to media at normal oxygenconditions resulted in a reproducible dose-dependent increase inabsorbance and thus the viable cell number. The addition of serum tocomplete MSCGM resulted in a dose-dependent decrease in absorbance. Inthe media without added serum, cells only grew appreciably inCELLGRO-FREE, Ham's F10 and DMEM.

The Effect of Oxygen. Reduced oxygen appeared to increase the growthrate of cells in Growth Medium, Ham's F10, and MSCGM. In decreasingorder of growth, the media resulting in the best growth of the cellswere Growth Medium>MSCGM>Iscove's+10% FBS=DMEM-H+10% FBS=Ham's F12+10%FBS═RPMI 1640+10% FBS.

Summary. Placenta-derived cells may be grown in a variety of culturemedia in normal or low oxygen. Short term growth of placenta-derivedcells was determined in twelve basal media with 0, 2 and 10% (v/v) serumin 5% or atmospheric oxygen. In general, placenta-derived cells did notgrow as well in serum-free conditions with the exception of Ham's F10and CELLGRO-Free, which are also protein-free. Growth in theseserum-free media was about 25-33% of the maximal growth observed withmedia containing 15% serum.

EXAMPLE 4 Growth of Postpartum-Derived Cells in Medium ContainingD-Valine

It has been reported that medium containing D-valine instead of thenormal L-valine isoform can be used to selectively inhibit the growth offibroblast-like cells in culture (Hongpaisan, 2000; Sordillo et al.,1988). It was not previously known whether postpartum-derived cellscould grow in medium containing D-valine.

Methods & Materials

Placenta-derived cells (P3), fibroblasts (P9) and umbilical-derivedcells (P5) were seeded at 5×10³ cells/cm² in gelatin-coated T75 flasks(Corning, Corning, N.Y.). After 24 hours the medium was removed and thecells were washed with phosphate buffered saline (PBS) (Gibco, Carlsbad,Calif.) to remove residual medium. The medium was replaced with aModified Growth Medium (DMEM with D-valine (special order Gibco), 15%(v/v) dialyzed fetal bovine serum (Hyclone, Logan, Utah), 0.001% (v/v)betamercaptoethanol (Sigma), penicillin/streptomycin (Gibco)).

Results

Placenta-derived, umbilical-derived, and fibroblast cells seeded in theD-valine-containing medium did not proliferate, unlike cells seeded inGrowth Medium containing dialyzed serum. Fibroblasts cells changedmorphologically, increasing in size and changing shape. All of the cellsdied and eventually detached from the flask surface after 4 weeks. Theseresults indicate that medium containing D-valine is not suitable forselectively growing postpartum-derived cells.

References for Example 4

1) Hongpaisan J. 2000. Cell Biol Int. 24:1-7.

2) Sordillo L M, Oliver S P, Akers R M. 1988). Cell Biol Int Rep.12:355-64.

EXAMPLE 5 Cryopreservation Media for Placenta-Derived Cells

Cryopreservation media for the cryopreservation of placenta-derivedcells were evaluated.

Methods & Materials

Placenta-derived cells grown in Growth Medium in a gelatin-coated T75flask were washed with PBS and trypsinized using 1 milliliterTrypsin/EDTA (Gibco). The trypsinization was stopped by adding 10milliliters Growth Medium. The cells were centrifuged at 150×g,supernatant removed, and the cell pellet was resuspended in 1 milliliterGrowth Medium. An aliquot of cell suspension, 60 microliters, wasremoved and added to 60 microliters trypan blue (Sigma). The viable cellnumber was estimated using a hemocytometer. The cell suspension wasdivided into four equal aliquots each containing 88×10⁴ cells each. Thecell suspension was centrifuged and resuspended in 1 milliliter of eachmedia below and transferred into Cryovials (Nalgene).

-   -   1.) Growth Medium+10% (v/v) DMSO (Hybrimax, Sigma, St. Louis,        Mo.)    -   2.) Cell Freezing medium w/DMSO, w/methyl cellulose, serum-free        (C6295, Sigma, St. Louis, Mo.)    -   3.) Cell Freezing medium serum-free (C2639, Sigma, St. Louis,        Mo.)    -   4.) Cell Freezing Medium w/glycerol (C6039, Sigma, St. Louis,        Mo.)

The cells were cooled at approximately −1° C./min overnight in a −80° C.freezer using a “Mr Frosty” freezing container according to themanufacturer's instructions (Nalgene, Rochester, N.Y.). Vials of cellswere transferred into liquid nitrogen for 2 days before thawing rapidlyin a 37° C. water bath. The cells were added to 10 milliliters GrowthMedium and centrifuged before the cell number and viability wasestimated. Cells were seeded onto gelatin-coated flasks at 5,000cells/cm² to determine whether the cells would attach and proliferate.

Results

The initial viability of the cells to be cryopreserved was assessed bytrypan blue staining to be 100%. The initial viability of the cells tobe cryopreserved was assessed by trypan blue staining to be 100%.

There was a commensurate reduction in cell number with viability forC6295 due to cells lysis. The viable cells cryopreserved in all foursolutions attached, divided, and produced a confluent monolayer within 3days. There was no discernable difference in estimated growth rate.

Summary. The cryopreservation of cells is one procedure available forpreparation of a cell bank or a cell product. Four cryopreservationmixtures were compared for their ability to protect humanplacenta-derived cells from freezing damage. Dulbecco's modified Eagle'smedium (DMEM) and 10% (v/v) dimethylsulfoxide (DMSO) is the preferredmedium of those compared for cryopreservation of placenta-derived cells.

EXAMPLE 6 Karyotype Analysis of Postpartum-Derived Cells

Cell lines used in cell therapy are preferably homogeneous and free fromany contaminating cell type. Cells used in cell therapy should have anormal chromosome number (46) and structure. To identify placenta- andumbilicus-derived cell lines that are homogeneous and free from cells ofnon-postpartum tissue origin, karyotypes of cell samples were analyzed.

Materials and Methods

PPDCs from postpartum tissue of a male neonate were cultured in GrowthMedium containing penicillin/streptomycin. Postpartum tissue from a maleneonate (X,Y) was selected to allow distinction between neonatal-derivedcells and maternal derived cells (X,X). Cells were seeded at 5,000 cellsper square centimeter in Growth Medium in a T25 flask (Corning, Corning,N.Y.) and expanded to 80% confluence. A T25 flask containing cells wasfilled to the neck with Growth Medium. Samples were delivered to aclinical cytogenetics laboratory by courier (estimated lab to labtransport time is one hour). Cells were analyzed during metaphase whenthe chromosomes are best visualized. Of twenty cells in metaphasecounted, five were analyzed for normal homogeneous karyotype number(two). A cell sample was characterized as homogeneous if two karyotypeswere observed. A cell sample was characterized as heterogeneous if morethan two karyotypes were observed. Additional metaphase cells werecounted and analyzed when a heterogeneous karyotype number (four) wasidentified.

Results

All cell samples sent for chromosome analysis were interpreted asexhibiting a normal appearance. Three of the sixteen cell lines analyzedexhibited a heterogeneous phenotype (XX and XY) indicating the presenceof cells derived from both neonatal and maternal origins (Table 6-1).Cells derived from tissue Placenta-N were isolated from the neonatalaspect of placenta. At passage zero, this cell line appeared homogeneousXY. However, at passage nine, the cell line was heterogeneous (XX/XY),indicating a previously undetected presence of cells of maternal origin.

TABLE 6-1 Results of PPDC karyotype analysis Metaphase cells Metaphasecells Number of Tissue passage counted analyzed karyotypes ISCNKaryotype Placenta 22 20 5 2 46, XX Umbilical 23 20 5 2 46, XX Umbilical6 20 5 2 46, XY Placenta 2 20 5 2 46, XX Umbilical 3 20 5 2 46, XXPlacenta-N 0 20 5 2 46, XY Placenta-V 0 20 5 2 46, XY Placenta-M 0 21 54 46, XY[18]/46, XX[3] Placenta-M 4 20 5 2 46, XX Placenta-N 9 25 5 446, XY[5]/46, XX[20] Placenta-N 1 20 5 2 46, XY C1 Placenta-N 1 20 6 446, XY[2]/46, C3 XX[18] Placenta-N 1 20 5 2 46, XY C4 Placenta-N 1 20 52 46, XY C15 Placenta-N 1 20 5 2 46, XY C20 Key: N- Neonatal aspect; V-villous region; M- maternal aspect; C- clone

Summary. Chromosome analysis identified placenta- and umbilicus-derivedcells whose karyotypes appeared normal as interpreted by a clinicalcytogenetic laboratory. Karyotype analysis also identified cell linesfree from maternal cells, as determined by homogeneous karyotype.

EXAMPLE 7 Evaluation of Human Postpartum-Derived Cell Surface Markers byFlow Cytometry

Characterization of cell surface proteins or “markers” by flow cytometrycan be used to determine a cell line's identity. The consistency ofexpression can be determined from multiple donors, and in cells exposedto different processing and culturing conditions. Postpartum-derivedcell (PPDC) lines isolated from the placenta and umbilicus werecharacterized (by flow cytometry), providing a profile for theidentification of these cell lines.

Materials and Methods

Media and culture vessels. Cells were cultured in Growth Medium (GibcoCarlsbad, Calif.) with penicillin/streptomycin. Cells were cultured inplasma-treated T75, T150, and T225 tissue culture flasks (Corning,Corning, N.Y.) until confluent. The growth surfaces of the flasks werecoated with gelatin by incubating 2% (w/v) gelatin (Sigma, St. Louis,Mo.) for 20 minutes at room temperature.

Antibody Staining and flow cytometry analysis. Adherent cells in flaskswere washed in PBS and detached with Trypsin/EDTA. Cells were harvested,centrifuged, and resuspended in 3% (v/v) FBS in PBS at a cellconcentration of 1×10⁷ per milliliter. In accordance to themanufacture's specifications, antibody to the cell surface marker ofinterest (see below) was added to one hundred microliters of cellsuspension and the mixture was incubated in the dark for 30 minutes at4° C. After incubation, cells were washed with PBS and centrifuged toremove unbound antibody. Cells were resuspended in 500 microliter PBSand analyzed by flow cytometry. Flow cytometry analysis was performedwith a FACScalibur instrument (Becton Dickinson, San Jose, Calif.).

The following antibodies to cell surface markers were used.

Catalog Antibody Manufacture Number CD10 BD Pharmingen (San Diego, CA)555375 CD13 BD Pharmingen 555394 CD31 BD Pharmingen 555446 CD34 BDPharmingen 555821 CD44 BD Pharmingen 555478 CD45RA BD Pharmingen 555489CD73 BD Pharmingen 550257 CD90 BD Pharmingen 555596 CD117 BD Pharmingen340529 CD141 BD Pharmingen 559781 PDGFr-alpha BD Pharmingen 556002HLA-A, B, C BD Pharmingen 555553 HLA-DR, DP, DQ BD Pharmingen 555558IgG-FITC Sigma (St. Louis, MO) F-6522 IgG-PE Sigma P-4685

Placenta and umbilicus comparison. Placenta-derived cells were comparedto umbilicus-derive cells at passage 8.

Passage to passage comparison. Placenta- and umbilicus-derived cellswere analyzed at passages 8, 15, and 20.

Donor to donor comparison. To compare differences among donors,placenta-derived cells from different donors were compared to eachother, and umbilicus-derived cells from different donors were comparedto each other.

Surface coating comparison. Placenta-derived cells cultured ongelatin-coated flasks was compared to placenta-derived cells cultured onuncoated flasks. Umbilicus-derived cells cultured on gelatin-coatedflasks was compared to umbilicus-derived cells cultured on uncoatedflasks.

Digestion enzyme comparison. Four treatments used for isolation andpreparation of cells were compared. Cells isolated from placenta bytreatment with 1) collagenase; 2) collagenase/dispase; 3)collagenase/hyaluronidase; and 4) collagenase/hyaluronidase/dispase werecompared.

Placental layer comparison. Cells derived from the maternal aspect ofplacental tissue were compared to cells derived from the villous regionof placental tissue and cells derived from the neonatal fetal aspect ofplacenta.

Results

Placenta vs. umbilicus comparison. Placenta- and umbilicus-derived cellsanalyzed by flow cytometry showed positive expression of CD10, CD13,CD44, CD73, CD 90, PDGFr-alpha and HLA-A, B, C, indicated by theincreased values of fluorescence relative to the IgG control. Thesecells were negative for detectable expression of CD31, CD34, CD45,CD117, CD141, and HLA-DR, DP, DQ, indicated by fluorescence valuescomparable to the IgG control. Variations in fluorescence values ofpositive curves were accounted for. The mean (i.e. CD13) and range (i.e.CD90) of the positive curves showed some variation, but the curvesappeared normal, confirming a homogenous population. Both curvesindividually exhibited values greater than the IgG control.

Passage to passage comparison—placenta-derived cells. Placenta-derivedcells at passages 8, 15, and 20 analyzed by flow cytometry all werepositive for expression of CD10, CD13, CD44, CD73, CD 90, PDGFr-alphaand HLA-A, B, C, as reflected in the increased value of fluorescencerelative to the IgG control. The cells were negative for expression ofCD31, CD34, CD45, CD117, CD141, and HLA-DR, DP, DQ having fluorescencevalues consistent with the IgG control.

Passage to passage comparison—umbilicus-derived cells. Umbilicus-derivedcells at passage 8, 15, and 20 analyzed by flow cytometry all expressedCD10, CD13, CD44, CD73, CD 90, PDGFr-alpha and HLA-A, B, C, indicated byincreased fluorescence relative to the IgG control. These cells werenegative for CD31, CD34, CD45, CD117, CD141, and HLA-DR, DP, DQ,indicated by fluorescence values consistent with the IgG control.

Donor to donor comparison—placenta-derived cells. Placenta-derived cellsisolated from separate donors analyzed by flow cytometry each expressedCD10, CD13, CD44, CD73, CD 90, PDGFr-alpha and HLA-A, B, C, withincreased values of fluorescence relative to the IgG control. The cellswere negative forexpression of CD31, CD34, CD45, CD117, CD141, andHLA-DR, DP, DQ as indicated by fluorescence value consistent with theIgG control.

Donor to donor comparison—umbilicus derived cells. Umbilicus-derivedcells isolated from separate donors analyzed by flow cytometry eachshowed positive expression of CD10, CD13, CD44, CD73, CD 90, PDGFr-alphaand HLA-A, B, C, reflected in the increased values of fluorescencerelative to the IgG control. These cells were negative for expression ofCD31, CD34, CD45, CD117, CD141, and HLA-DR, DP, DQ with fluorescencevalues consistent with the IgG control.

The effect of surface coating with gelatin on placenta-derived cells.Placenta-derived cells expanded on either gelatin-coated or uncoatedflasks analyzed by flow cytometry all expressed of CD10, CD13, CD44,CD73, CD 90, PDGFr-alpha and HLA-A, B, C, reflected in the increasedvalues of fluorescence relative to the IgG control. These cells werenegative for expression of CD31, CD34, CD45, CD117, CD141, and HLA-DR,DP, DQ indicated by fluorescence values consistent with the IgG control.

The effect of surface coating with gelatin on umbilicus-derived cells.Umbilicus-derived cells expanded on gelatin and uncoated flasks analyzedby flow cytometry all were positive for expression of CD10, CD13, CD44,CD73, CD 90, PDGFr-alpha and HLA-A, B, C, with increased values offluorescence relative to the IgG control. These cells were negative forexpression of CD31, CD34, CD45, CD117, CD141, and HLA-DR, DP, DQ, withfluorescence values consistent with the IgG control.

Effect of enzyme digestion procedure used for preparation of the cellson the cell surface marker profile. Placenta-derived cells isolatedusing various digestion enzymes analyzed by flow cytometry all expressedCD10, CD13, CD44, CD73, CD 90, PDGFr-alpha and HLA-A, B, C, as indicatedby the increased values of fluorescence relative to the IgG control.These cells were negative for expression of CD31, CD34, CD45, CD117,CD141, and HLA-DR, DP, DQ as indicated by fluorescence values consistentwith the IgG control.

Placental layer comparison. Cells isolated from the maternal, villous,and neonatal layers of the placenta, respectively, analyzed by flowcytometry showed positive expression of CD10, CD13, CD44, CD73, CD 90,PDGFr-alpha and HLA-A, B, C, as indicated by the increased value offluorescence relative to the IgG control. These cells were negative forexpression of CD31, CD34, CD45, CD117, CD141, and HLA-DR, DP, DQ asindicated by fluorescence values consistent with the IgG control.

Summary. Analysis of placenta- and umbilicus-derived cells by flowcytometry has established of an identity of these cell lines. Placenta-and umbilicus-derived cells are positive for CD10, CD13, CD44, CD73,CD90, PDGFr-alpha, HLA-A,B,C and negative for CD31, CD34, CD45, CD117,CD141 and HLA-DR, DP, DQ. This identity was consistent betweenvariations in variables including the donor, passage, culture vesselsurface coating, digestion enzymes, and placental layer. Some variationin individual fluorescence value histogram curve means and ranges wasobserved, but all positive curves under all conditions tested werenormal and expressed fluorescence values greater than the IgG control,thus confirming that the cells comprise a homogenous population that haspositive expression of the markers.

EXAMPLE 8 Immunohistochemical Characterization of Postpartum TissuePhenotypes

The phenotypes of cells found within human postpartum tissues, namelyumbilical cord and placenta, was analyzed by immunohistochemistry.

Materials & Methods

Tissue Preparation. Human umbilical cord and placenta tissue washarvested and immersion fixed in 4% (w/v) paraformaldehyde overnight at4° C. Immunohistochemistry was performed using antibodies directedagainst the following epitopes: vimentin (1:500; Sigma, St. Louis, Mo.),desmin (1:150, raised against rabbit; Sigma; or 1:300, raised againstmouse; Chemicon, Temecula, Calif.), alpha-smooth muscle actin (SMA;1:400; Sigma), cytokeratin 18 (CK18; 1:400; Sigma), von WillebrandFactor (vWF; 1:200; Sigma), and CD34 (human CD34 Class III; 1:100;DAKOCytomation, Carpinteria, Calif.). In addition, the following markerswere tested: anti-human GROalpha-PE (1:100; Becton Dickinson, FranklinLakes, N.J.), anti-human GCP-2 (1:100; Santa Cruz Biotech, Santa Cruz,Calif.), anti-human oxidized LDL receptor 1 (ox-LDL R1; 1:100; SantaCruz Biotech), and anti-human NOGO-A (1:100; Santa Cruz Biotech). Fixedspecimens were trimmed with a scalpel and placed within OCT embeddingcompound (Tissue-Tek OCT; Sakura, Torrance, Calif.) on a dry ice bathcontaining ethanol. Frozen blocks were then sectioned (10 μm thick)using a standard cryostat (Leica Microsystems) and mounted onto glassslides for staining.

Immunohistochemistry. Immunohistochemistry was performed similar toprevious studies (e.g., Messina, et al., 2003, Exper. Neurol. 184:816-829). Tissue sections were washed with phosphate-buffered saline(PBS) and exposed to a protein blocking solution containing PBS, 4%(v/v) goat serum (Chemicon, Temecula, Calif.), and 0.3% (v/v) Triton(Triton X-100; Sigma) for 1 hour to access intracellular antigens. Ininstances where the epitope of interest would be located on the cellsurface (CD34, ox-LDL R1), Triton was omitted in all steps of theprocedure in order to prevent epitope loss. Furthermore, in instanceswhere the primary antibody was raised against goat (GCP-2, ox-LDL R1,NOGO-A), 3% (v/v) donkey serum was used in place of goat serumthroughout the procedure. Primary antibodies, diluted in blockingsolution, were then applied to the sections for a period of 4 hours atroom temperature. Primary antibody solutions were removed, and cultureswashed with PBS prior to application of secondary antibody solutions (1hour at room temperature) containing block along with goat anti-mouseIgG—Texas Red (1:250; Molecular Probes, Eugene, Oreg.) and/or goatanti-rabbit IgG—Alexa 488 (1:250; Molecular Probes) or donkey anti-goatIgG—FITC (1: 150; Santa Cruz Biotech). Cultures were washed, and 10micromolar DAPI (Molecular Probes) was applied for 10 minutes tovisualize cell nuclei.

Following immunostaining, fluorescence was visualized using theappropriate fluorescence filter on an Olympus inverted epi-fluorescentmicroscope (Olympus, Melville, N.Y.). Positive staining was representedby fluorescence signal above control staining. Representative imageswere captured using a digital color videocamera and ImagePro software(Media Cybernetics, Carlsbad, Calif.). For triple-stained samples, eachimage was taken using only one emission filter at a time. Layeredmontages were then prepared using Adobe Photoshop software (Adobe, SanJose, Calif.).

Results

Umbilical cord characterization. Vimentin, desmin, SMA, CK18, vWF, andCD34 markers were expressed in a subset of the cells found withinumbilical cord. In particular, vWF and CD34 expression were restrictedto blood vessels contained within the cord. CD34+cells were on theinnermost layer (lumen side). Vimentin expression was found throughoutthe matrix and blood vessels of the cord. SMA was limited to the matrixand outer walls of the artery & vein, but not contained with the vesselsthemselves. CK18 and desmin were observed within the vessels only,desmin being restricted to the middle and outer layers.

Placenta characterization. Vimentin, desmin, SMA, CK18, vWF, and CD34were all observed within the placenta and regionally specific.

GROalpha, GCP-2, ox-LDL R1, and NOGO-A Tissue Expression. None of thesemarkers were observed within umbilical cord or placental tissue.

Summary. Vimentin, desmin, alpha-smooth muscle actin, cytokeratin 18,von Willebrand Factor, and CD 34 are expressed in cells within humanumbilical cord and placenta.

EXAMPLE 9 Analysis of Postpartum Tissue-Derived Cells usingOligonucleotide Arrays

Affymetrix GENECHIP arrays were used to compare gene expression profilesof umbilicus- and placenta-derived cells with fibroblasts, humanmesenchymal stem cells, and another cell line derived from human bonemarrow. This analysis provided a characterization of thepostpartum-derived cells and identified unique molecular markers forthese cells.

Materials and Methods

Isolation and culture of cells. Human umbilical cords and placenta wereobtained from National Disease Research Interchange (NDRI, Philadelphia,Pa.) from normal full term deliveries with patient consent. The tissueswere received and cells were isolated as described in Example 1. Cellswere cultured in Growth Medium (using DMEM-LG) on gelatin-coated tissueculture plastic flasks. The cultures were incubated at 37° C. with 5%CO₂.

Human dermal fibroblasts were purchased from Cambrex Incorporated(Walkersville, Md.; Lot number 9F0844) and ATCC CRL-1501 (CCD39SK). Bothlines were cultured in DMEM/F12 medium (Invitrogen, Carlsbad, Calif.)with 10% (v/v) fetal bovine serum (Hyclone) and penicillin/streptomycin(Invitrogen). The cells were grown on standard tissue-treated plastic.

Human mesenchymal stem cells (hMSC) were purchased from CambrexIncorporated (Walkersville, Md.; Lot numbers 2F1655, 2F1656 and 2F1657)and cultured according to the manufacturer's specifications in MSCGMMedia (Cambrex). The cells were grown on standard tissue culturedplastic at 37° C. with 5% CO₂.

Human iliac crest bone marrow was received from NDR1 with patientconsent. The marrow was processed according to the method outlined byHo, et al. (WO03/025149). The marrow was mixed with lysis buffer (155 mMNH₄Cl, 10 mM KHCO₃, and 0.1 mM EDTA, pH 7.2) at a ratio of 1 part bonemarrow to 20 parts lysis buffer. The cell suspension was vortexed,incubated for 2 minutes at ambient temperature, and centrifuged for 10minutes at 500×g. The supernatant was discarded and the cell pellet wasresuspended in Minimal Essential Medium-alpha (Invitrogen) supplementedwith 10% (v/v) fetal bovine serum and 4 mM glutamine. The cells werecentrifuged again and the cell pellet was resuspended in fresh medium.The viable mononuclear cells were counted using trypan-blue exclusion(Sigma, St. Louis, Mo.). The mononuclear cells were seeded intissue-cultured plastic flasks at 5×104 cells/cm². The cells wereincubated at 37° C. with 5% CO₂ at either standard atmospheric O₂ or at5% O₂. Cells were cultured for 5 days without a media change. Media andnon-adherent cells were removed after 5 days of culture. The adherentcells were maintained in culture.

Isolation of mRNA and GENECHIP Analysis. Actively growing cultures ofcells were removed from the flasks with a cell scraper in cold PBS. Thecells were centrifuged for 5 minutes at 300×g. The supernatant wasremoved and the cells were resuspended in fresh PBS and centrifugedagain. The supernatant was removed and the cell pellet was immediatelyfrozen and stored at −80° C. Cellular mRNA was extracted and transcribedinto cDNA, which was then transcribed into cRNA and biotin-labeled. Thebiotin-labeled cRNA was hybridized with HG-U133A GENECHIPoligonucleotide array (Affymetrix, Santa Clara Calif.). Thehybridization and data collection was performed according to themanufacturer's specifications. Analyses were performed using“Significance Analysis of Microarrays” (SAM) version 1.21 computersoftware (Stanford University; Tusher, V. G. et al., 2001, Proc. Natl.Acad. Sci. USA 98: 5116-5121).

Results

Fourteen different populations of cells were analyzed. The cells alongwith passage information, culture substrate, and culture media arelisted in Table 9-1.

TABLE 9-1 Cells analyzed by the microarray study. Cell lines are listedby identification code along with passage at time of analysis, cellgrowth substrate and growth medium. Cell Population Passage SubstrateMedium Umbilicus (022803) 2 Gelatin DMEM, 15% FBS, 2-ME Umbilicus(042103) 3 Gelatin DMEM, 15% FBS, 2-ME Umbilicus (071003) 4 GelatinDMEM, 15% FBS, 2-ME Placenta (042203) 12 Gelatin DMEM, 15% FBS, 2-MEPlacenta (042903) 4 Gelatin DMEM, 15% FBS, 2-ME Placenta (071003) 3Gelatin DMEM, 15% FBS, 2-ME ICBM (070203) (5% 02) 3 Plastic MEM, 10% FBSICBM (062703) (std. O2) 5 Plastic MEM, 10% FBS ICBM (062703) (5% 02) 5Plastic MEM, 10% FBS hMSC (Lot 2F1655) 3 Plastic MSCGM hMSC (Lot 2F1656)3 Plastic MSCGM hMSC (Lot 2F 1657) 3 Plastic MSCGM hFibroblast (9F0844)9 Plastic DMEM-F12, 10% FBS hFibroblast (CCD39SK) 4 Plastic DMEM-F12,10% FBS

The data were evaluated by a Principle Component Analysis, analyzing the290 genes that were differentially expressed in the cells. This analysisallows for a relative comparison for the similarities between thepopulations. Table 9-2 shows the Euclidean distances that werecalculated for the comparison of the cell pairs. The Euclidean distanceswere based on the comparison of the cells based on the 290 genes thatwere differentially expressed among the cell types. The Euclideandistance is inversely proportional to similarity between the expressionof the 290 genes (i.e., the greater the distance, the less similarityexists).

TABLE 9-2 The Euclidean Distances for the Cell Pairs. Cell PairEuclidean Distance ICBM-hMSC 24.71 Placenta-umbilical 25.52ICBM-Fibroblast 36.44 ICBM-placenta 37.09 Fibroblast-MSC 39.63ICBM-Umbilical 40.15 Fibroblast-Umbilical 41.59 MSC-Placenta 42.84MSC-Umbilical 46.86 ICBM-placenta 48.41

Tables 9-3,9-4, and 9-5 show the expression of genes increased inplacenta-derived cells (Table 9-3), increased in umbilicus-derived cells(Table 9-4), and reduced in umbilicus- and placenta-derived cells (Table9-5). The column entitled “Probe Set ID” refers to the manufacturer'sidentification code for the sets of several oligonucleotide probeslocated on a particular site on the chip, which hybridize to the namedgene (column “Gene Name”), comprising a sequence that can be foundwithin the NCBI (GenBank) database at the specified accession number(column “NCBI Accession Number”).

TABLE 9-3 Genes shown to have specifically increased expression in theplacenta-derived cells as compared to other cell lines assayed GenesIncreased in Placenta-Derived Cells NCBI Probe Accession Set ID GeneName Number 209732_at C-type (calcium dependent, AF070642carbohydrate-recognition domain) lectin, superfamily member 2(activation-induced) 206067_s_at Wilms tumor 1 NM_024426 207016_s_ataldehyde dehydrogenase 1 AB015228 family, member A2 206367_at reninNM_000537 210004_at oxidized low density AF035776 lipoprotein(lectin-like) receptor 1 214993_at Homo sapiens, clone IMAGE: AF0706424179671, mRNA, partial cds 202178_at protein kinase C, zeta NM_002744209780_at hypothetical protein AL136883 DKFZp564F013 204135_atdownregulated in ovarian NM_014890 cancer 1 213542_at Homo sapiens mRNA;cDNA AI246730 DKFZp547K1113 (from clone DKFZp547K1113)

TABLE 9-4 Genes shown to have specifically increased expression in theumbilicus-derived cells as compared to other cell lines assayed GenesIncreased in Umbilicus-Derived Cells NCBI Probe Set Accession ID GeneName Number 202859_x_at interleukin 8 NM_000584 211506_s_at interleukin8 AF043337 210222_s_at reticulon 1 BC000314 204470_at chemokine (C-X-Cmotif) ligand 1 NM_001511 (melanoma growth stimulating activity206336_at chemokine (C-X-C motif) ligand 6 NM_002993 (granulocytechemotactic protein 2) 207850_at chemokine (C-X-C motif) ligand 3NM_002090 203485_at reticulon 1 NM_021136 202644_s_at tumor necrosisfactor, alpha-induced NM_006290 protein 3

TABLE 9-5 Genes shown to have decreased expression in umbilicus- andplacenta-derived cells as compared to other cell lines assayed GenesDecreased in Umbilicus- and Placenta-Derived Cells NCBI Probe SetAccession ID Gene name Number 210135_s_at short stature homeobox 2AF022654.1 205824_at heat shock 27 kDa protein 2 NM_001541.1 209687_atchemokine (C-X-C motif) ligand 12 U19495.1 (stromal cell-derivedfactor 1) 203666_at chemokine (C-X-C motif) ligand 12 NM_000609.1(stromal cell-derived factor 1) 212670_at elastin (supravalvular aorticAA479278 stenosis, Williams-Beuren syndrome) 213381_at Homo sapiensmRNA; cDNA N91149 DKFZp586M2022 (from clone DKFZp586M2022) 206201_s_atmesenchyme homeobox 2 (growth NM_005924.1 arrest-specific homeo box)205817_at sine oculis homeobox homolog 1 NM_005982.1 (Drosophila)209283_at crystallin, alpha B AF007162.1 212793_at dishevelledassociated activator BF513244 of morphogenesis 2 213488_at DKFZP586B2420protein AL050143.1 209763_at similar to neuralin 1 AL049176 205200_attetranectin (plasminogen binding NM_003278.1 protein) 205743_at srchomology three (SH3) and NM_003149.1 cysteine rich domain 200921_s_atB-cell translocation gene 1, NM_001731.1 anti-proliferative 206932_atcholesterol 25-hydroxylase NM_003956.1 204198_s_at runt-relatedtranscription AA541630 factor 3 219747_at hypothetical protein FLJ23191NM_024574.1 204773_at interleukin 11 receptor, alpha NM_004512.1202465_at procollagen C-endopeptidase NM_002593.2 enhancer 203706_s_atfrizzled homolog 7 (Drosophila) NM_003507.1 212736_at hypothetical geneBC008967 BE299456 214587_at collagen, type VIII, alpha 1 BE877796201645_at tenascin C (hexabrachion) NM_002160.1 210239_at iroquoishomeobox protein 5 U90304.1 203903_s_at hephaestin NM_014799.1 205816_atintegrin, beta 8 NM_002214.1 203069_at synaptic vesicle glycoprotein 2NM_014849.1 213909_at Homo sapiens cDNA FLJ12280 fis, AU147799 cloneMAMMA1001744 206315_at cytokine receptor-like factor 1 NM_004750.1204401_at potassium intermediate/small NM_002250.1 conductancecalcium-activated channel, subfamily N, member 4 216331_at integrin,alpha 7 AK022548.1 209663_s_at integrin, alpha 7 AF072132.1 213125_atDKFZP586L151 protein AW007573 202133_at transcriptional co-activatorAA081084 with PDZ-binding motif (TAZ) 206511_s_at sine oculis homeoboxhomolog 2 NM_016932.1 (Drosophila) 213435_at KIAA1034 protein AB028957.1206115_at early growth response 3 NM_004430.1 213707_s_at distal-lesshomeo box 5 NM_005221.3 218181_s_at hypothetical protein FLJ20373NM_017792.1 209160_at aldo-keto reductase family 1, AB018580.1 member C3(3-alpha hydroxysteroid dehydrogenase, type II) 213905_x_at biglycanAA845258 201261_x_at biglycan BC002416.1 202132_at transcriptionalco-activator AA081084 with PDZ-binding motif (TAZ) 214701_s_atfibronectin 1 AJ276395.1 213791_at proenkephalin NM_006211.1 205422_s_atintegrin, beta-like 1 (with NM_004791.1 EGF-like repeat domains)214927_at Homo sapiens mRNA full length AL359052.1 insert cDNA cloneEUROIMAGE 1968422 206070_s_at EphA3 AF213459.1 212805_at KIAA0367protein AB002365.1 219789_at natriuretic peptide receptor AI628360C/guanylate cyclase C (atrionatriuretic peptide receptor C) 219054_athypothetical protein FLJ14054 NM_024563.1 213429_at Homo sapiens mRNA;cDNA AW025579 DKFZp564B222 (from clone DKFZp564B222) 204929_s_atvesicle-associated membrane NM_006634.1 protein 5 (myobrevin)201843_s_at EGF-containing fibulin-like NM_004105.2 extracellular matrixprotein 1 221478_at BCL2/adenovirus E1B 19 kDa AL132665.1 interactingprotein 3-like 201792_at AE binding protein 1 NM_001129.2 204570_atcytochrome c oxidase subunit NM_001864.1 VIIa polypeptide 1 (muscle)201621_at neuroblastoma, suppression of NM_005380.1 tumorigenicity 1202718_at insulin-like growth factor NM_000597.1 binding protein 2, 36kDa

Tables 9-6,9-7, and 9-8 show the expression of genes increased in humanfibroblasts (Table 9-6), ICBM cells (Table 9-7), and MSCs (Table 9-8).

TABLE 9-6 Genes that were shown to have increased expression infibroblasts as compared to the other cell lines assayed. Genes increasedin fibroblasts dual specificity phosphatase 2 KIAA0527 protein Homosapiens cDNA: FLJ23224 fis, clone ADSU02206 dynein, cytoplasmic,intermediate polypeptide 1 ankyrin 3, node of Ranvier (ankyrin G)inhibin, beta A (activin A, activin AB alpha polypeptide) ectonucleotidepyrophosphatase/phosphodiesterase 4 (putative function) KIAA1053 proteinmicrotubule-associated protein 1A zinc finger protein 41 HSPC019 proteinHomo sapiens cDNA: FLJ23564 fis, clone LNG10773 Homo sapiens mRNA; cDNADKFZp564A072 (from clone DKFZp564A072) LIM protein (similar to ratprotein kinase C-binding enigma) inhibitor of kappa light polypeptidegene enhancer in B-cells, kinase complex-associated protein hypotheticalprotein FLJ22004 Human (clone CTG-A4) mRNA sequence ESTs, Moderatelysimilar to cytokine receptor-like factor 2; cytokine receptor CRL2precursor [Homo sapiens] transforming growth factor, beta 2 hypotheticalprotein MGC29643 antigen identified by monoclonal antibody MRC OX-2putative X-linked retinopathy protein

TABLE 9-7 Genes that were shown to have increased expression in theICBM-derived cells as compared to the other cell lines assayed. GenesIncreased In ICBM Cells cardiac ankyrin repeat protein MHC class Iregion ORF integrin, alpha 10 hypothetical protein FLJ22362UDP-N-acetyl-alpha-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase 3 (GalNAc-T3) interferon-induced protein44 SRY (sex determining region Y)-box 9 (campomelic dysplasia, autosomalsex-reversal) keratin associated protein 1-1 hippocalcin-like 1 jagged 1(Alagille syndrome) proteoglycan 1, secretory granule

TABLE 9-8 Genes that were shown to have increased expression in the MSCcells as compared to the other cell lines assayed. Genes Increased InMSC Cells interleukin 26 maltase-glucoamylase (alpha-glucosidase)nuclear receptor subfamily 4, group A, member 2 v-fos FBJ murineosteosarcoma viral oncogene homolog hypothetical protein DC42 nuclearreceptor subfamily 4, group A, member 2 FBJ murine osteosarcoma viraloncogene homolog B WNT1 inducible signaling pathway protein 1 MCF.2 cellline derived transforming sequence potassium channel, subfamily K,member 15 cartilage paired-class homeoprotein 1 Homo sapiens cDNAFLJ12232 fis, clone MAMMA1001206 Homo sapiens cDNA FLJ34668 fis, cloneLIVER2000775 jun B proto-oncogene B-cell CLL/lymphoma 6 (zinc fingerprotein 51) zinc finger protein 36, C3H type, homolog (mouse)

Summary. The present examination was performed to provide a molecularcharacterization of the postpartum cells derived from umbilical cord andplacenta. This analysis included cells derived from three differentumbilical cords and three different placentas. The examination alsoincluded two different lines of dermal fibroblasts, three lines ofmesenchymal stem cells, and three lines of iliac crest bone marrowcells. The mRNA that was expressed by these cells was analyzed using anoligonucleotide array that contained probes for 22,000 genes. Resultsshowed that 290 genes are differentially expressed in these fivedifferent cell types. These genes include ten genes that arespecifically increased in the placenta-derived cells and seven genesspecifically increased in the umbilical cord-derived cells. Fifty-fourgenes were found to have specifically lower expression levels inplacenta and umbilical cord, as compared with the other cell types. Theexpression of selected genes has been confirmed by PCR (see the examplethat follows). These results demonstrate that the postpartum-derivedcells have a distinct gene expression profile, for example, as comparedto bone marrow-derived cells and fibroblasts.

EXAMPLE 10 Cell Markers in Postpartum-Derived Cells

In the preceding example, similarities and differences in cells derivedfrom the human placenta and the human umbilical cord were assessed bycomparing their gene expression profiles with those of cells derivedfrom other sources (using an oligonucleotide array). Six “signature”genes were identified: oxidized LDL receptor 1, interleukin-8, rennin,reticulon, chemokine receptor ligand 3 (CXC ligand 3), and granulocytechemotactic protein 2 (GCP-2). These “signature” genes were expressed atrelatively high levels in postpartum-derived cells.

The procedures described in this example were conducted to verify themicroarray data and find concordance/discordance between gene andprotein expression, as well as to establish a series of reliable assayfor detection of unique identifiers for placenta- and umbilicus-derivedcells.

Methods & Materials

Cells. Placenta-derived cells (three isolates, including one isolatepredominately neonatal as identified by karyotyping analysis),umbilicus-derived cells (four isolates), and Normal Human DermalFibroblasts (NHDF; neonatal and adult) grown in Growth Medium withpenicillin/streptomycin in a gelatin-coated T75 flask. Mesechymal StemCells (MSCs) were grown in Mesenchymal Stem Cell Growth Medium Bulletkit (MSCGM; Cambrex, Walkerville, Md.).

For the IL-8 protocol, cells were thawed from liquid nitrogen and platedin gelatin-coated flasks at 5,000 cells/cm², grown for 48 hours inGrowth Medium and then grown for further 8 hours in 10 milliliters ofserum starvation medium [DMEM-low glucose (Gibco, Carlsbad, Calif.),penicillin/streptomycin (Gibco, Carlsbad, Calif.) and 0.1% (w/v) BovineSerum Albumin (BSA; Sigma, St. Louis, Mo.)]. After this treatment RNAwas extracted and the supernatants were centrifuged at 150×g for 5minutes to remove cellular debris. Supernatants were then frozen at −80°C. for ELISA analysis.

Cell culture for ELISA assay. Postpartum cells derived from placenta andumbilicus, as well as human fibroblasts derived from human neonatalforeskin were cultured in Growth Medium in gelatin-coated T75 flasks.Cells were frozen at passage 11 in liquid nitrogen. Cells were thawedand transferred to 15-milliliter centrifuge tubes. After centrifugationat 150×g for 5 minutes, the supernatant was discarded. Cells wereresuspended in 4 milliliters culture medium and counted. Cells weregrown in a 75 cm² flask containing 15 milliliters of Growth Medium at375,000 cell/flask for 24 hours. The medium was changed to a serumstarvation medium for 8 hours. Serum starvation medium was collected atthe end of incubation, centrifuged at 14,000×g for 5 minutes (and storedat −20° C.).

To estimate the number of cells in each flask, 2 milliliters oftyrpsin/EDTA (Gibco, Carlsbad, Calif.) was added each flask. After cellsdetached from the flask, trypsin activity was neutralized with 8milliliters of Growth Medium. Cells were transferred to a 15 milliliterscentrifuge tube and centrifuged at 150×g for 5 minutes. Supernatant wasremoved and 1 milliliter Growth Medium was added to each tube toresuspend the cells. Cell number was estimated using a hemocytometer.

ELISA assay. The amount of IL-8 secreted by the cells into serumstarvation medium was analyzed using ELISA assays (R&D Systems,Minneapolis, Minn.). All assays were tested according to theinstructions provided by the manufacturer.

Total RNA isolation. RNA was extracted from confluent postpartum-derivedcells and fibroblasts or for IL-8 expression from cells treated asdescribed above. Cells were lysed with 350 microliters buffer RLTcontaining beta-mercaptoethanol (Sigma, St. Louis, Mo.) according to themanufacturer's instructions (RNeasy Mini Kit; Qiagen, Valencia, Calif.).RNA was extracted according to the manufacturer's instructions (RNeasyMini Kit; Qiagen, Valencia, Calif.) and subjected to DNase treatment(2.7 U/sample) (Sigma St. Louis, Mo.). RNA was eluted with 50microliters DEPC-treated water and stored at −80° C.

Reverse transcription. RNA was also extracted from human placenta andumbilicus. Tissue (30 milligram) was suspended in 700 microliters ofbuffer RLT containing 2-mercaptoethanol. Samples were mechanicallyhomogenized and the RNA extraction proceeded according to manufacturer'sspecification. RNA was extracted with 50 microliters of DEPC-treatedwater and stored at −80° C. RNA was reversed transcribed using randomhexamers with the TaqMan reverse transcription reagents (AppliedBiosystems, Foster City, Calif.) at 25° C. for 10 minutes, 37° C. for 60minutes, and 95° C. for 10 minutes. Samples were stored at −20° C.

Genes identified by cDNA microarray as uniquely regulated in postpartumcells (signature genes—including oxidized LDL receptor, interleukin-8,rennin and reticulon), were further investigated using real-time andconventional PCR.

Real-time PCR. PCR was performed on cDNA samples using Assays-on-Demand™gene expression products: oxidized LDL receptor (Hs00234028); rennin(Hs00166915); reticulon (Hs00382515); CXC ligand 3 (Hs00171061); GCP-2(Hs00605742); IL-8 (Hs00174103); and GAPDH (Applied Biosystems, FosterCity, Calif.) were mixed with cDNA and TaqMan Universal PCR master mixaccording to the manufacturer's instructions (Applied Biosystems, FosterCity, Calif.) using a 7000 sequence detection system with ABI Prism 7000SDS software (Applied Biosystems, Foster City, Calif.). Thermal cycleconditions were initially 50° C. for 2 min and 95° C. for 10 min,followed by 40 cycles of 95° C. for 15 sec and 60° C. for 1 min. PCRdata was analyzed according to manufacturer's specifications (UserBulletin #2 from Applied Biosystems for ABI Prism 7700 SequenceDetection System).

Conventional PCR. Conventional PCR was performed using an ABI PRISM 7700(Perkin Elmer Applied Biosystems, Boston, Mass., USA) to confirm theresults from real-time PCR. PCR was performed using 2 microliters ofcDNA solution, 1×AmpliTaq Gold universal mix PCR reaction buffer(Applied Biosystems, Foster City, Calif.) and initial denaturation at94° C. for 5 minutes. Amplification was optimized for each primer set.For IL-8, CXC ligand 3, and reticulon (94° C. for 15 seconds, 55° C. for15 seconds and 72° C. for 30 seconds for 30 cycles); for rennin (94° C.for 15 seconds, 53° C. for 15 seconds and 72° C. for 30 seconds for 38cycles); for oxidized LDL receptor and GAPDH (94° C. for 15 seconds, 55°C. for 15 seconds and 72° C. for 30 seconds for 33 cycles). Primers usedfor amplification are listed in Table 1. Primer concentration in thefinal PCR reaction was 1 micromolar except for GAPDH, which was 0.5micromolar. GAPDH primers were the same as real-time PCR, except thatthe manufacturer's TaqMan probe was not added to the final PCR reaction.Samples were run on 2% (w/v) agarose gel and stained with ethidiumbromide (Sigma, St. Louis, Mo.). Images were captured using a 667Universal Twinpack film (VWR International, South Plainfield, N.J.)using a focal-length Polaroid camera (VWR International, SouthPlainfield, N.J.).

TABLE 10-1 Primers used Primer name Primers Oxidized LDL S:5′-GAGAAATCCAAAGAGCAAATGG-3′ (SEQ ID NO:1) receptor A:5′-AGAATGGAAAACTGGAATAGG-3′ (SEQ ID NO:2) Renin S:5′-TCTTCGATGCTTCGGATTCC-3′ (SEQ ID NO:3) A: 5′-GAATTCTCGGAATCTCTGTTG-3′(SEQ ID NO:4) Reticulon S: 5′-TTACAAGCAGTGCAGAAAACC-3′ (SEQ ID NO:5) A:5′-AGTAAACATTGAAACCACAGCC-3′ (SEQ ID NO:6) Interleukin-8 S:5′-TCTGCAGCTCTGTGTGAAGG-3′ (SEQ ID NO:7) A: 5′-CTTCAAAAACTTCTCCACAACC-3′(SEQ ID NO:8) Chemokine (CXC) S: 5′-CCCACGCCACGCTCTCC-3′ (SEQ ID NO:9)ligand 3 A: 5′-TCCTGTCAGTTGGTGCTCC-3′ (SEQ ID NO:10)

Immunofluorescence. PPDCs were fixed with cold 4% (w/v) paraformaldehyde(Sigma-Aldrich, St. Louis, Mo.) for 10 minutes at room temperature. Oneisolate each of umbilicus- and placenta-derived cells at passage 0 (P0)(directly after isolation) and passage 11 (P11) (two isolates ofplacenta-derived, two isolates of umbilicus-derived cells) andfibroblasts (P11) were used. Immunocytochemistry was performed usingantibodies directed against the following epitopes: vimentin (1:500,Sigma, St. Louis, Mo.), desmin (1:150; Sigma—raised against rabbit; or1:300; Chemicon, Temecula, Calif.—raised against mouse,), alpha-smoothmuscle actin (SMA; 1:400; Sigma), cytokeratin 18 (CK18; 1:400; Sigma),von Willebrand Factor (vWF; 1:200; Sigma), and CD34 (human CD34 ClassIII; 1:100; DAKOCytomation, Carpinteria, Calif.). In addition, thefollowing markers were tested on passage 11 postpartum cells: anti-humanGRO alpha—PE (1:100; Becton Dickinson, Franklin Lakes, N.J.), anti-humanGCP-2 (1:100; Santa Cruz Biotech, Santa Cruz, Calif.), anti-humanoxidized LDL receptor 1 (ox-LDL R1; 1:100; Santa Cruz Biotech), andanti-human NOGA-A (1:100; Santa Cruz, Biotech).

Cultures were washed with phosphate-buffered saline (PBS) and exposed toa protein blocking solution containing PBS, 4% (v/v) goat serum(Chemicon, Temecula, Calif.), and 0.3% (v/v) Triton (Triton X-100;Sigma, St. Louis, Mo.) for 30 minutes to access intracellular antigens.Where the epitope of interest was located on the cell surface (CD34,ox-LDL R1), Triton X-100 was omitted in all steps of the procedure inorder to prevent epitope loss. Furthermore, in instances where theprimary antibody was raised against goat (GCP-2, ox-LDL R1, NOGO-A), 3%(v/v) donkey serum was used in place of goat serum throughout. Primaryantibodies, diluted in blocking solution, were then applied to thecultures for a period of 1 hour at room temperature. The primaryantibody solutions were removed and the cultures were washed with PBSprior to application of secondary antibody solutions (1 hour at roomtemperature) containing block along with goat anti-mouse IgG—Texas Red(1:250; Molecular Probes, Eugene, Oreg.) and/or goat anti-rabbitIgG—Alexa 488 (1:250; Molecular Probes) or donkey anti-goat IgG—FITC(1:150, Santa Cruz Biotech). Cultures were then washed and 10 micromolarDAPI (Molecular Probes) applied for 10 minutes to visualize cell nuclei.

Following immunostaining, fluorescence was visualized using anappropriate fluorescence filter on an Olympus inverted epi-fluorescentmicroscope (Olympus, Melville, N.Y.). In all cases, positive stainingrepresented fluorescence signal above control staining where the entireprocedure outlined above was followed with the exception of applicationof a primary antibody solution. Representative images were capturedusing a digital color videocamera and ImagePro software (MediaCybernetics, Carlsbad, Calif.). For triple-stained samples, each imagewas taken using only one emission filter at a time. Layered montageswere then prepared using Adobe Photoshop software (Adobe, San Jose,Calif.).

Preparation of cells for FACS analysis. Adherent cells in flasks werewashed in phosphate buffered saline (PBS) (Gibco, Carlsbad, Calif.) anddetached with Trypsin/EDTA (Gibco, Carlsbad, Calif.). Cells wereharvested, centrifuged, and re-suspended 3% (v/v) FBS in PBS at a cellconcentration of 1×10⁷ per milliliter. One hundred microliter aliquotswere delivered to conical tubes. Cells stained for intracellularantigens were permeablized with Perm/Wash buffer (BD Pharmingen, SanDiego, Calif.). Antibody was added to aliquots as per manufacturesspecifications and the cells were incubated for in the dark for 30minutes at 4° C. After incubation, cells were washed with PBS andcentrifuged to remove excess antibody. Cells requiring a secondaryantibody were resuspended in 100 microliters of 3% FBS. Secondaryantibody was added as per manufactures specification and the cells wereincubated in the dark for 30 minutes at 4° C. After incubation, cellswere washed with PBS and centrifuged to remove excess secondaryantibody. Washed cells were resuspended in 0.5 milliliters PBS andanalyzed by flow cytometry. The following antibodies were used: oxidizedLDL receptor 1 (sc-5813; Santa Cruz, Biotech), GROa (555042; BDPharmingen, Bedford, Mass.), Mouse IgG1 kappa, (P-4685 and M-5284;Sigma), Donkey against Goat IgG (sc-3743; Santa Cruz, Biotech.). Flowcytometry analysis was performed with FACScalibur (Becton Dickinson SanJose, Calif.).

Results

Results of real-time PCR for selected “signature” genes performed oncDNA from cells derived from human placentae, adult and neonatalfibroblasts and Mesenchymal Stem Cells (MSCs) indicate that bothoxidized LDL receptor and rennin were expressed at higher level in theplacenta-derived cells as compared to other cells. The data obtainedfrom real-time PCR were analyzed by the AACT method and expressed on alogarithmic scale. Levels of reticulon and oxidized LDL receptorexpression were higher in umbilicus-derived cells as compared to othercells. No significant difference in the expression levels of CXC ligand3 and GCP-2 were found between postpartum-derived cells and controls.The results of real-time PCR were confirmed by conventional PCR.Sequencing of PCR products further validated these observations. Nosignificant difference in the expression level of CXC ligand 3 was foundbetween postpartum-derived cells and controls using conventional PCR CXCligand 3 primers listed above.

The production of the cytokine, IL-8 in postpartum was elevated in bothGrowth Medium-cultured and serum-starved postpartum-derived cells. Allreal-time PCR data was validated with conventional PCR and by sequencingPCR products.

When supernatants of cells grown in serum-free medium were examined forthe presence of IL-8, the highest amounts were detected in media derivedfrom umbilical cells and some isolates of placenta cells (Table 10-1).No IL-8 was detected in medium derived from human dermal fibroblasts.

TABLE 10-1 IL-8 protein amount measured by ELISA Cell type IL-8 hFibroND Placenta Isolate 1 ND Umb Isolate 1 2058.42 ± 144.67 Placenta Isolate2 ND Umb Isolate 2 2368.86 ± 22.73 Placenta Isolate3 (normal O₂) 17.27 ±8.63 Placenta Isolate 3 (low O₂, W/O BME) 264.92 ± 9.88 Results of theELISA assay for interleukin-8 (IL-8) performed on placenta- andumbilicus-derived cells as well as human skin fibroblasts. Values arepresented here are picograms/million cells, n = 2, sem. ND: Not Detected

Placenta-derived cells were also examined for the production of oxidizedLDL receptor, GCP-2 and GROalpha by FACS analysis. Cells tested positivefor GCP-2. Oxidized LDL receptor and GRO were not detected by thismethod.

Placenta-derived cells were also tested for the production of selectedproteins by immunocytochernical analysis. Immediately after isolation(passage 0), cells derived from the human placenta were fixed with 4%paraformaldehyde and exposed to antibodies for six proteins: vonWillebrand Factor, CD34, cytokeratin 18, desmin, alpha-smooth muscleactin, and vimentin. Cells stained positive for both alpha-smooth muscleactin and vimentin. This pattern was preserved through passage 11. Onlya few cells (<5%) at passage 0 stained positive for cytokeratin 18.

Cells derived from the human umbilical cord at passage 0 were probed forthe production of selected proteins by immunocytochemical analysis.Immediately after isolation (passage 0), cells were fixed with 4%paraformaldehyde and exposed to antibodies for six proteins: vonWillebrand Factor, CD34, cytokeratin 18, desmin, alpha-smooth muscleactin, and vimentin. Umbilicus-derived cells were positive foralpha-smooth muscle actin and vimentin, with the staining patternconsistent through passage 11.

Summary. Concordance between gene expression levels measured bymicroarray and PCR (both real-time and conventional) has beenestablished for four genes: oxidized LDL receptor 1, rennin, reticulon,and IL-8. The expression of these genes was differentially regulated atthe mRNA level in PPDCs, with IL-8 also differentially regulated at theprotein level. The presence of oxidized LDL receptor was not detected atthe protein level by FACS analysis in cells derived from the placenta.Differential expression of GCP-2 and CXC ligand 3 was not confirmed atthe mRNA level, however GCP-2 was detected at the protein level by FACSanalysis in the placenta-derived cells. Although this result is notreflected by data originally obtained from the microarray experiment,this may be due to a difference in the sensitivity of the methodologies.

Immediately after isolation (passage 0), cells derived from the humanplacenta stained positive for both alpha-smooth muscle actin andvimentin. This pattern was also observed in cells at passage 11. Theseresults suggest that vimentin and alpha-smooth muscle actin expressionmay be preserved in cells with passaging, in the Growth Medium and underthe conditions utilized in these procedures. Cells derived from thehuman umbilical cord at passage 0 were probed for the expression ofalpha-smooth muscle actin and vimentin, and were positive for both. Thestaining pattern was preserved through passage 11.

EXAMPLE 11 In Vitro Immunological Evaluation of Postpartum-Derived Cells

Postpartum-derived cells (PPDCs) were evaluated in vitro for theirimmunological characteristics in an effort to predict the immunologicalresponse, if any, these cells would elicit upon in vivo transplantation.PPDCs were assayed by flow cytometry for the presence of HLA-DR, HLA-DP,HLA-DQ, CD80, CD86, and B7-H2. These proteins are expressed byantigen-presenting cells (APC) and are required for the directstimulation of naïve CD4⁺ T cells (Abbas & Lichtman, CELLULAR ANDMOLECULAR IMMUNOLOGY, 5th Ed. (2003) Saunders, Philadelphia, p. 171).The cell lines were also analyzed by flow cytometry for the expressionof HLA-G (Abbas & Lichtman, 2003, supra), CD 178 (Coumans, et al.,(1999) Journal of Immunological Methods 224, 185-196), and PD-L2 (Abbas& Lichtman, 2003, supra; Brown, et. al. (2003) The Journal of Immunology170, 1257-1266). The expression of these proteins by cells residing inplacental tissues is thought to mediate the immuno-privileged status ofplacental tissues in utero. To predict the extent to which placenta- andumbilicus-derived cell lines elicit an immune response in vivo, the celllines were tested in a one-way mixed lymphocyte reaction (MLR).

Materials and Methods

Cell culture. Cells were cultured to confluence in Growth Mediumcontaining penicillin/streptomycin in T75 flasks (Corning, Corning,N.Y.) coated with 2% gelatin (Sigma, St. Louis, Mo.).

Antibody Staining. Cells were washed in phosphate buffered saline (PBS)(Gibco, Carlsbad, Calif.) and detached with Trypsin/EDTA (Gibco,Carlsbad, Mo.). Cells were harvested, centrifuged, and re-suspended in3% (v/v) FBS in PBS at a cell concentration of 1×10⁷ per milliliter.Antibody (Table 11-1) was added to one hundred microliters of cellsuspension as per manufacturer's specifications and incubated in thedark for 30 minutes at 4° C. After incubation, cells were washed withPBS and centrifuged to remove unbound antibody. Cells were re-suspendedin five hundred microliters of PBS and analyzed by flow cytometry usinga FACSCalibur instrument (Becton Dickinson, San Jose, Calif.).

TABLE 11-1 Antibodies Catalog Antibody Manufacturer Number HLA-DRDPDQ BDPharmingen (San Diego, CA) 555558 CD80 BD Pharmingen (San Diego, CA)557227 CD86 BD Pharmingen (San Diego, CA) 555665 B7-H2 BD Pharmingen(San Diego, CA) 552502 HLA-G Abcam (Cambridgeshire, UK) ab 7904-100 CD178 Santa Cruz (San Cruz, CA) sc-19681 PD-L2 BD Pharmingen (San Diego,CA) 557846 Mouse IgG2a Sigma (St. Louis, MO) F-6522 Mouse IgG1kappaSigma (St. Louis, MO) P-4685

Mixed Lymphocyte Reaction. Cryopreserved vials of passage 10umbilicus-derived cells labeled as cell line A and passage 11placenta-derived cells labeled as cell line B were sent on dry ice toCTBR (Senneville, Quebec) to conduct a mixed lymphocyte reaction usingCTBR SOP No. CAC-031. Peripheral blood mononuclear cells (PBMCs) werecollected from multiple male and female volunteer donors. Stimulator(donor) allogeneic PBMC, autologous PBMC, and postpartum cell lines weretreated with mitomycin C. Autologous and mitomycin C-treated stimulatorcells were added to responder (recipient) PBMCs and cultured for 4 days.After incubation, [³H]thymidine was added to each sample and culturedfor 18 hours. Following harvest of the cells, radiolabeled DNA wasextracted, and [³H]-thymidine incorporation was measured using ascintillation counter.

The stimulation index for the allogeneic donor (SIAD) was calculated asthe mean proliferation of the receiver plus mitomycin C-treatedallogeneic donor divided by the baseline proliferation of the receiver.The stimulation index of the PPDCs was calculated as the meanproliferation of the receiver plus mitomycin C-treated postpartum cellline divided by the baseline proliferation of the receiver.

Results

Mixed lymphocyte reaction—placenta-derived cells. Seven human volunteerblood donors were screened to identify a single allogeneic donor thatwould exhibit a robust proliferation response in a mixed lymphocytereaction with the other six blood donors. This donor was selected as theallogeneic positive control donor. The remaining six blood donors wereselected as recipients. The allogeneic positive control donor andplacenta-derived cell lines were treated with mitomycin C and culturedin a mixed lymphocyte reaction with the six individual allogeneicreceivers. Reactions were performed in triplicate using two cell cultureplates with three receivers per plate (Table 11-2). The averagestimulation index ranged from 1.3 (plate 2) to 3 (plate 1) and theallogeneic donor positive controls ranged from 46.25 (plate 2) to 279(plate 1) (Table 11-3).

TABLE 11-2 Mixed Lymphocyte Reaction Data - Cell Line B (Placenta) DPMfor Proliferation Assay Analytical Culture Replicates number System 1 23 Mean SD CV Plate ID: Plate1 IM03-7769 Proliferation baseline ofreceiver 79 119 138 112.0 30.12 26.9 Control of autostimulation(Mitomycin C treated autologous cells) 241 272 175 229.3 49.54 21.6 MLRallogenic donor IM03-7768 (Mitomycin C treated) 23971 22352 2092122414.7 1525.97 6.8 MLR with cell line (Mitomycin C treated cell type B)664 559 1090 771.0 281.21 36.5 SI (donor) 200 SI (cell line) 7 IM03-7770Proliferation baseline of receiver 206 134 262 200.7 64.17 32.0 Controlof autostimulation (Mitomycin C treated autologous cells) 1091 602 524739.0 307.33 41.6 MLR allogenic donor IM03-7768 (Mitomycin C treated)45005 43729 44071 44268.3 660.49 1.5 MLR with cell line (Mitomycin Ctreated cell type B) 533 2582 2376 1830.3 1128.24 61.6 SI (donor) 221 SI(cell line) 9 IM03-7771 Proliferation baseline of receiver 157 87 128124.0 35.17 28.4 Control of autostimulation (Mitomycin C treatedautologous cells) 293 138 508 313.0 185.81 59.4 MLR allogenic donorIM03-7768 (Mitomycin C treated) 24497 34348 31388 30077.7 5054.53 16.8MLR with cell line (Mitomycin C treated cell type B) 601 643 a 622.029.70 4.8 SI (donor) 243 SI (cell line) 5 IM03-7772 Proliferationbaseline of receiver 56 98 51 68.3 25.81 37.8 Control of autostimulation(Mitomycin C treated autologous cells) 133 120 213 155.3 50.36 32.4 MLRallogenic donor IM03-7768 (Mitomycin C treated) 14222 20076 2216818822.0 4118.75 21.9 MLR with cell line (Mitomycin C treated cell typeB) a a a a a a SI (donor) 275 SI (cell line) a IM03-7768 Proliferationbaseline of receiver 84 242 208 178.0 83.16 46.7 (allogenic donor)Control of autostimulation (Mitomycin treated autologous cells) 361 617304 427.3 166.71 39.0 Cell line type B Proliferation baseline ofreceiver 126 124 143 131.0 10.44 8.0 Control of autostimulation(Mitomycin treated autologous cells) 822 1075 487 794.7 294.95 37.1Plate ID: Plate 2 IM03-7773 Proliferation baseline of receiver 908 181330 473.0 384.02 81.2 Control of autostimulation (Mitomycin C treatedautologous cells) 269 405 572 415.3 151.76 36.5 MLR allogenic donorIM03-7768 (Mitomycin C treated) 29151 28691 28315 28719.0 418.70 1.5 MLRwith cell line (Mitomycin C treated cell type B) 567 732 905 734.7169.02 23.0 SI (donor) 61 SI (cell line) 2 IM03-7774 Proliferationbaseline of receiver 893 1376 185 818.0 599.03 73.2 Control ofautostimulation (Mitomycin C treated autologous cells) 261 381 568 403.3154.71 38.4 MLR allogenic donor IM03-7768 (Mitomycin C treated) 5310142839 48283 48074.3 5134.18 10.7 MLR with cell line (Mitomycin C treatedcell type B) 515 789 294 532.7 247.97 46.6 SI (donor) 59 SI (cell line)1 IM03-7775 Proliferation baseline of receiver 1272 300 544 705.3 505.6971.7 Control of autostimulation (Mitomycin C treated autologous cells)232 199 484 305.0 155.89 51.1 MLR allogenic donor IM03-7768 (Mitomycin Ctreated) 23554 10523 28965 21014.0 9479.74 45.1 MLR with cell line(Mitomycin C treated cell type B) 768 924 563 751.7 181.05 24.1 SI(donor) 30 SI (cell line) 1 IM03-7776 Proliferation baseline of receiver1530 137 1046 904.3 707.22 78.2 Control of autostimulation (Mitomycin Ctreated autologous cells) 420 218 394 344.0 109.89 31.9 MLR allogenicdonor IM03-7768 (Mitomycin C treated) 28893 32493 34746 32044.0 2952.229.2 MLR with cell line (Mitomycin C treated cell type B) a a a a a a SI(donor) 35 SI (cell line) a

TABLE 11-3 Average stimulation index of placenta cells and an allogeneicdonor in a mixed lymphocyte reaction with six individual allogeneicreceivers Average Stimulation Index Recipient Placenta Plate 1(receivers 1-3) 279 3 Plate 2 (receivers 4-6) 46.25 1.3

Mixed lymphocyte reaction—umbilicus-derived cells. Six human volunteerblood donors were screened to identify a single allogeneic donor thatwill exhibit a robust proliferation response in a mixed lymphocytereaction with the other five blood donors. This donor was selected asthe allogeneic positive control donor. The remaining five blood donorswere selected as recipients. The allogeneic positive control donor andplacenta cell lines were mitomycin C-treated and cultured in a mixedlymphocyte reaction with the five individual allogeneic receivers.Reactions were performed in triplicate using two cell culture plateswith three receivers per plate (Table 11-4). The average stimulationindex ranged from 6.5 (plate 1) to 9 (plate 2) and the allogeneic donorpositive controls ranged from 42.75 (plate 1) to 70 (plate 2) (Table11-5).

TABLE 11-4 Mixed Lymphocyte Reaction Data- Cell Line A (Umbilicus) DPMfor Proliferation Assay Analytical Culture Replicates number System 1 23 Mean SD CV Plate ID: Plate1 IM04-2478 Proliferation baseline ofreceiver 1074 406 391 623.7 390.07 62.5 Control of autostimulation(Mitomycin C treated autologous cells) 672 510 1402 861.3 475.19 55.2MLR allogenic donor IM04-2477 (Mitomycin C treated) 43777 48391 3823143466.3 5087.12 11.7 MLR with cell line (Mitomycin C treated cell typeA) 2914 5622 6109 4881.7 1721.36 35.3 SI (donor) 70 SI (cell line) 8IM04-2479 Proliferation baseline of receiver 530 508 527 521.7 11.93 2.3Control of autostimulation (Mitomycin C treated autologous cells) 701567 1111 793.0 283.43 35.7 MLR allogenic donor IM04-2477 (Mitomycin Ctreated) 25593 24732 22707 24344.0 1481.61 6.1 MLR with cell line(Mitomycin C treated cell type A) 5086 3932 1497 3505.0 1832.21 52.3 SI(donor) 47 SI (cell line) 7 IM04-2480 Proliferation baseline of receiver1192 854 1330 1125.3 244.90 21.8 Control of autostimulation (Mitomycin Ctreated autologous cells) 2963 993 2197 2051.0 993.08 48.4 MLR allogenicdonor IM04-2477 (Mitomycin C treated) 25416 29721 23757 26298.0 3078.2711.7 MLR with cell line (Mitomycin C treated cell type A) 2596 5076 34263699.3 1262.39 34.1 SI (donor) 23 SI (cell line) 3 IM04-2481Proliferation baseline of receiver 695 451 555 567.0 122.44 21.6 Controlof autostimulation (Mitomycin C treated autologous cells) 738 1252 464818.0 400.04 48.9 MLR allogenic donor IM04-2477 (Mitomycin C treated)13177 24885 15444 17835.3 6209.52 34.8 MLR with cell line (Mitomycin Ctreated cell type A) 4495 3671 4674 4280.0 534.95 12.5 SI (donor) 31 SI(cell line) 8 Plate ID: Plate 2 IM04-2482 Proliferation baseline ofreceiver 432 533 274 413.0 130.54 31.6 Control of autostimulation(Mitomycin C treated autologous cells) 1459 633 598 896.7 487.31 54.3MLR allogenic donor IM04-2477 (Mitomycin C treated) 24286 30823 3134628818.3 3933.82 13.7 MLR with cell line (Mitomycin C treated cell typeA) 2762 1502 6723 3662.3 2724.46 74.4 SI (donor) 70 SI (cell line) 9IM04-2477 Proliferation baseline of receiver 312 419 349 360.0 54.3415.1 (allogenic donor) Control of autostimulation (Mitomycin treatedautologous cells) 567 604 374 515.0 123.50 24.0 Cell line type AProliferation baseline of receiver 5101 3735 2973 3936.3 1078.19 27.4Control of autostimulation (Mitomycin treated autologous cells) 19244570 2153 2882.3 1466.04 50.9

TABLE 11-5 Average stimulation index of umbilicus-derived cells and anallogeneic donor in a mixed lymphocyte reaction with five individualallogeneic receivers. Average Stimulation Index Recipient UmbilicusPlate 1 (receivers 1-4) 42.75 6.5 Plate 2 (receiver 5) 70 9

Antigen presenting cell markers—placenta-derived cells. Histograms ofplacenta-derived cells analyzed by flow cytometry show negativeexpression of HLA-DR, DP, DQ, CD80, CD86, and B7-H2, as noted byfluorescence value consistent with the IgG control, indicating thatplacental cell lines lack the cell surface molecules required todirectly stimulate CD4⁺ T cells.

Immunomodulating markers—placenta-derived cells. Histograms ofplacenta-derived cells analyzed by flow cytometry show positiveexpression of PD-L2, as noted by the increased value of fluorescencerelative to the IgG control, and negative expression of CD178 and HLA-G,as noted by fluorescence value consistent with the IgG control.

Antigen presenting cell markers—umbilicus-derived cells. Histograms ofumbilicus-derived cells analyzed by flow cytometry show negativeexpression of HLA-DR, DP, DQ, CD80, CD86, and B7-H2, as noted byfluorescence value consistent with the IgG control, indicating thatumbilical cell lines lack the cell surface molecules required todirectly stimulate CD4⁺ T cells.

Immunomodulating cell markers—umbilicus-derived cells. Histograms ofumbilicus-derived cells analyzed by flow cytometry show positiveexpression of PD-L2, as noted by the increased value of fluorescencerelative to the IgG control, and negative expression of CD178 and HLA-G,as noted by fluorescence value consistent with the IgG control.

Summary. In the mixed lymphocyte reactions conducted withplacenta-derived cell lines, the average stimulation index ranged from1.3 to 3, and that of the allogeneic positive controls ranged from 46.25to 279. In the mixed lymphocyte reactions conducted withumbilicus-derived cell lines the average stimulation index ranged from6.5 to 9, and that of the allogeneic positive controls ranged from 42.75to 70. Placenta- and umbilicus-derived cell lines were negative for theexpression of the stimulating proteins HLA-DR, HLA-DP, HLA-DQ, CD80,CD86, and B7-H2, as measured by flow cytometry. Placenta- andumbilicus-derived cell lines were negative for the expression ofimmuno-modulating proteins HLA-G and CD 178 and positive for theexpression of PD-L2, as measured by flow cytometry. Allogeneic donorPBMCs contain antigen-presenting cells expressing HLA-DR, DQ, CD8, CD86,and B7-H2, thereby allowing for the stimulation of naïve CD4⁺ T cells.The absence of antigen-presenting cell surface molecules on placenta-and umbilicus-derived cells required for the direct stimulation of naïveCD4⁺ T cells and the presence of PD-L2, an immunomodulating protein, mayaccount for the low stimulation index exhibited by these cells in a MLRas compared to allogeneic controls.

EXAMPLE 12 Secretion of Trophic Factors by Postpartum-Derived Cells

The secretion of selected trophic factors from placenta- andumbilicus-derived cells was measured. Factors selected for detectionincluded: (1) those known to have angiogenic activity, such ashepatocyte growth factor (HGF) (Rosen et al. (1997) Ciba Found. Symp.212:215-26), monocyte chemotactic protein 1 (MCP-1) (Salcedo et al.(2000) Blood 96;34-40), interleukin-8 (IL-8) (Li et al. (2003) J.Immunol. 170:3369-76), keratinocyte growth factor (KGF), basicfibroblast growth factor (bFGF), vascular endothelial growth factor(VEGF) (Hughes et al. (2004) Ann. Thorac. Surg. 77:812-8), matrixmetalloproteinase 1 (TIMP1), angiopoietin 2 (ANG2), platelet derivedgrowth factor (PDGF-bb), thrombopoietin (TPO), heparin-binding epidermalgrowth factor (HB-EGF), stromal-derived factor 1alpha (SDF-1alpha); (2)those known to have neurotrophic/neuroprotective activity, such asbrain-derived neurotrophic factor (BDNF) (Cheng et al. (2003) Dev. Biol.258;319-33), interleukin-6 (IL-6), granulocyte chemotactic protein-2(GCP-2), transforming growth factor beta2 (TGFbeta2); and (3) thoseknown to have chemokine activity, such as macrophage inflammatoryprotein 1alpha (MIP1a), macrophage inflammatory protein 1beta (MIP1b),monocyte chemoattractant-1 (MCP-1), Rantes (regulated on activation,normal T cell expressed and secreted), 1309, thymus andactivation-regulated chemokine (TARC), Eotaxin, macrophage-derivedchemokine (MDC), IL-8).

Methods & Materials

Cell culture. PPDCs from placenta and umbilicus as well as humanfibroblasts derived from human neonatal foreskin were cultured in GrowthMedium with penicillin/streptomycin on gelatin-coated T75 flasks. Cellswere cryopreserved at passage 11 and stored in liquid nitrogen. Afterthawing of the cells, Growth Medium was added to the cells followed bytransfer to a 15 milliliter centrifuge tube and centrifugation of thecells at 150×g for 5 minutes. The supernatant was discarded. The cellpellet was resuspended in 4 milliliters Growth Medium, and cells werecounted. Cells were seeded at 375,000 cells/75 cm² flask containing 15milliliters of Growth Medium and cultured for 24 hours. The medium waschanged to a serum-free medium (DMEM-low glucose (Gibco), 0.1% (w/v)bovine serum albumin (Sigma), penicillin/streptomycin (Gibco)) for 8hours. Conditioned serum-free medium was collected at the end ofincubation by centrifugation at 14,000×g for 5 minutes and stored at−20° C. To estimate the number of cells in each flask, cells were washedwith PBS and detached using 2 milliliters trypsin/EDTA. Trypsin activitywas inhibited by addition of 8 milliliters Growth Medium. Cells werecentrifuged at 150×g for 5 minutes. Supernatant was removed, and cellswere resuspended in 1 milliliter Growth Medium. Cell number wasestimated using a hemocytometer.

ELISA assay. Cells were grown at 37° C. in 5% carbon dioxide andatmospheric oxygen. Placenta-derived cells (batch 101503) also weregrown in 5% oxygen or beta-mercaptoethanol (BME). The amount of MCP-1,IL-6, VEGF, SDF-1alpha, GCP-2, IL-8, and TGF-beta 2 produced by eachcell sample was measured by an ELISA assay (R&D Systems, Minneapolis,Minn.). All assays were performed according to the manufacturer'sinstructions.

SearchLight multiplexed ELISA assay. Chemokines (MIP1a, MIP1b, MCP-1,Rantes, 1309, TARC, Eotaxin, MDC, IL8), BDNF, and angiogenic factors(HGF, KGF, bFGF, VEGF, TIMP1, ANG2, PDGF-bb, TPO, HB-EGF were measuredusing SearchLight Proteome Arrays (Pierce Biotechnology Inc.). TheProteome Arrays are multiplexed sandwich ELISAs for the quantitativemeasurement of two to 16 proteins per well. The arrays are produced byspotting a 2×2, 3×3, or 4×4 pattern of four to 16 different captureantibodies into each well of a 96-well plate. Following a sandwich ELISAprocedure, the entire plate is imaged to capture chemiluminescent signalgenerated at each spot within each well of the plate. The amount ofsignal generated in each spot is proportional to the amount of targetprotein in the original standard or sample.

Results

ELISA assay. MCP-1 and IL-6 were secreted by placenta- andumbilicus-derived cells and dermal fibroblasts (Table 12-1). SDF-1alphawas secreted by placenta-derived cells cultured in 5% O₂ and byfibroblasts. GCP-2 and IL-8 were secreted by umbilicus-derived cells andby placenta-derived cells cultured in the presence of BME or 5% O₂.GCP-2 also was secreted by human fibroblasts. TGF-beta2 was notdetectable by ELISA assay.

TABLE 12-1 ELISA assay results MCP-1 IL-6 VEGF SDF-1α GCP-2 IL-8 TGF-β2Fibroblast 17 ± 1 61 ± 3 29 ± 2 19 ± 1 21 ± 1 ND ND Placenta 60 ± 3 41 ±2 ND ND ND ND ND (042303) Umbilicus 1150 ± 74 4234 ± 289 ND ND 160 ± 112058 ± 145 ND (022803) Placenta 125 ± 16 10 ± 1 ND ND ND ND ND (071003)Umbilicus 2794 ± 84 1356 ± 43 ND ND 2184 ± 98 2369 ± 23 ND (071003)Placenta 21 ± 10 67 ± 3 ND ND 44 ± 9 17 ± 9 ND (101503) BME Placenta 77± 16 339 ± 21 ND 1149 ± 137 54 ± 2 265 ± 10 ND (101503) 5% O_(2,) W/OBME (values presented are picograms/milliliter/million cells (n = 2,sem) Key: ND: Not Detected.

SearchLight multiplexed ELISA assay. TIMP1, TPO, KGF, HGF, FGF, HBEGF,BDNF, MiP1b, MCP1, RANTES, I309, TARC, MDC, and IL-8 were secreted fromumbilicus-derived cells (Tables 12-2 and 12-3). TIMP1, TPO, KGF, HGF,HBEGF, BDNF, Mipla, MCP-1, RANTES, TARC, Eotaxin, and IL-8 were secretedfrom placenta-derived cells (Tables 12-2 and 12-3). No Ang2, VEGF, orPDGF-bb were detected.

TABLE 12-2 SearchLight Multiplexed ELISA assay results TIMP1 ANG2 PDGFbbTPO KGF HGF FGF VEGF HBEGF BDNF Hfb 19306.3 ND ND 230.5 5.0 ND ND 27.91.3 ND P1 24299.5 ND ND 546.6 8.8 16.4 ND ND 3.81.3 ND U1 57718.4 ND ND1240.0 5.8 559.3 148.7 ND 9.3 165.7 P3 14176.8 ND ND 568.7 5.2 10.2 NDND 1.9 33.6 U3 21850.0 ND ND 1134.5 9.0 195.6 30.8 ND 5.4 388.6 Key: hFB(human fibroblasts), P1 (placenta-derived cells (042303)), U1(umbilicus-derived cells (022803)), P3 (placenta-derived cells(071003)),U3 (umbilicus-derived cells (071003)). ND: Not Detected.

TABLE 12-3 SearchLight Multiplexed ELISA assay results MIP1a MIP1b MCP1RANTES I309 TARC Eotaxin MDC IL8 hFB ND ND 39.6 ND ND 0.1 ND ND 204.9 P179.5 ND 228.4 4.1 ND 3.8 12.2 ND 413.5 U1 ND 8.0 1694.2 ND 22.4 37.6 ND18.9 51930.1 P3 ND ND 102.7 ND ND 0.4 ND ND 63.8 U3 ND 5.2 2018.7 41.511.6 21.4 ND 4.8 10515.9 Key: hFB (human fibroblasts), P1(placenta-derived PPDC (042303)), U1 (umbilicus-derived PPDC (022803)),P3 (placenta-derived PPDC (071003)), U3 (umbilicus-derived PPDC(071003)). ND: Not Detected.

Summary. Umbilicus- and placenta-derived cells secreted a number oftrophic factors. Some of these trophic factors, such as HGF, bFGF, MCP-1and IL-8, play important roles in angiogenesis. Other trophic factors,such as BDNF and IL-6, have important roles in neural regeneration.

EXAMPLE 13 Short-Term Neural Differentiation of Postpartum-Derived Cells

The ability of placenta- and umbilicus-derived cells (collectivelypostpartum-derived cells or PPDCs) to differentiate into neural lineagecells was examined.

Materials & Methods

Isolation and Expansion of Postpartum Cells. PPDCs from placental andumbilical tissues were isolated and expanded as described in Example 1.

Modified Woodbury-Black Protocol. (A) This assay was adapted from anassay originally performed to test the neural induction potential ofbone marrow stromal cells (1). Umbilicus-derived cells (022803) P4 andplacenta-derived cells (042203) P3 were thawed and culture expanded inGrowth Media at 5,000 cells/cm² until sub-confluence (75%) was reached.Cells were then trypsinized and seeded at 6,000 cells per well of aTitretek II glass slide (VWR International, Bristol, Conn.). Ascontrols, mesenchymal stem cells (P3; 1F2155; Cambrex, Walkersville,Md.), osteoblasts (P5; CC2538; Cambrex), adipose-derived cells (Artecel,U.S. Pat. No. 6,555,374 B1) (P6; Donor 2) and neonatal human dermalfibroblasts (P6; CC2509; Cambrex) were also seeded under the sameconditions.

All cells were initially expanded for 4 days in DMEM/F12 medium(Invitrogen, Carlsbad, Calif.) containing 15% (v/v) fetal bovine serum(FBS; Hyclone, Logan, Utah), basic fibroblast growth factor (bFGF; 20nanograms/milliliter; Peprotech, Rocky Hill, N.J.), epidermal growthfactor (EGF; 20 nanograms/milliliter; Peprotech) andpenicillin/streptomycin (Invitrogen). After four days, cells were rinsedin phosphate-buffered saline (PBS; Invitrogen) and were subsequentlycultured in DMEM/F12 medium+20% (v/v) FBS+penicillin/streptomycin for 24hours. After 24 hours, cells were rinsed with PBS. Cells were thencultured for 1-6 hours in an induction medium which was comprised ofDMEM/F12 (serum-free) containing 200 mM butylated hydroxyanisole, 10 μMpotassium chloride, 5 milligram/milliliter insulin, 10 μM forskolin, 4μM valproic acid, and 2 μM hydrocortisone (all chemicals from Sigma, St.Louis, Mo.). Cells were then fixed in 100% ice-cold methanol andimmunocytochemistry was performed (see methods below) to assess humannestin protein expression.

(B) PPDCs (umbilicus (022803) P11; placenta (042203) P11) and adulthuman dermal fibroblasts (1F1853, P11) were thawed and culture expandedin Growth Medium at 5,000 cells/cm² until sub-confluence (75%) wasreached. Cells were then trypsinized and seeded at similar density as in(A), but onto (1) 24 well tissue culture-treated plates (TCP, Falconbrand, VWR International), (2) TCP wells+2% (w/v) gelatin adsorbed for 1hour at room temperature, or (3) TCP wells+20 μg/milliliter adsorbedmouse laminin (adsorbed for a minimum of 2 hours at 37° C.; Invitrogen).

Exactly as in (A), cells were initially expanded and media switched atthe aforementioned timeframes. One set of cultures was fixed, as before,at 5 days and six hours, this time with ice-cold 4% (w/v)paraformaldehyde (Sigma) for 10 minutes at room temperature. In thesecond set of cultures, medium was removed and switched to NeuralProgenitor Expansion medium (NPE) consisting of Neurobasal-A medium(Invitrogen) containing B27 (B27 supplement; Invitrogen), L-glutamine (4mM), and penicillin/streptomycin (Invitrogen). NPE medium was furthersupplemented with retinoic acid (RA; 1 μM; Sigma). This medium wasremoved 4 days later and cultures were fixed with ice-cold 4% (w/v)paraformaldehyde (Sigma) for 10 minutes at room temperature, and stainedfor nestin, GFAP, and TuJ1 protein expression (see Table N1-1).

TABLE 13-1 Summary of Primary Antibodies Used Antibody ConcentrationVendor Rat 401 (nestin) 1:200 Chemicon, Temecula, CA Human Nestin 1:100Chemicon TuJ1 (BIII Tubulin) 1:500 Sigma, St. Louis, MO GFAP 1:2000DakoCytomation, Carpinteria, CA Tyrosine 1:1000 Chemicon hydroxylase(TH) GABA 1:400 Chemicon Desmin (mouse) 1:300 Chemicon alpha -alpha-smooth 1:400 Sigma muscle actin Human nuclear 1:150 Chemiconprotein (hNuc)

Two Stage Differentiation Protocol. PPDCs (umbilicus (042203) P11,placenta (022803) P11), adult human dermal fibroblasts (P11; 1F1853;Cambrex) were thawed and culture expanded in Growth Medium at 5,000cells/cm² until sub-confluence (75%) was reached. Cells were thentrypsinized and seeded at 2,000 cells/cm², but onto 24 well platescoated with laminin (BD Biosciences, Franklin Lakes, N.J.) in thepresence of NPE media supplemented with bFGF (20 nanograms/milliliter;Peprotech, Rocky Hill, N.J.) and EGF (20 nanograms/milliliter;Peprotech) [whole media composition further referred to as NPE+F+E]. Atthe same time, adult rat neural progenitors isolated from hippocampus(P4; (062603) were also plated onto 24 well laminin-coated plates inNPE+F+E media. All cultures were maintained in such conditions for aperiod of 6 days (cells were fed once during that time) at which timemedia was switched to the differentiation conditions listed in TableN1-2 for an additional period of 7 days. Cultures were fixed withice-cold 4% (w/v) paraformaldehyde (sigma) for 10 minutes at roomtemperature, and stained for human or rat nestin, GFAP, and TuJ1 proteinexpression.

TABLE 13-2 Summary of Conditions for Two-Stage Differentiation ProtocolA B COND. # PRE-DIFFERENTIATION 2^(nd) STAGE DIFF 1 NPE + F (20 ng/ml) +E (20 ng/ml) NPE + SHH (200 ng/ml) + F8 (100 ng/ml) 2 NPE + F (20ng/ml) + E (20 ng/ml) NPE + SHH (200 ng/ml) + F8 (100 ng/ml) + RA (1 μM)3 NPE + F (20 ng/ml) + E (20 ng/ml) NPE + RA (1 μM) 4 NPE + F (20ng/ml) + E (20 ng/ml) NPE + F (20 ng/ml) + E (20 ng/ml) 5 NPE + F (20ng/ml) + E (20 ng/ml) Growth Medium 6 NPE + F (20 ng/ml) + E (20 ng/ml)Condition 1B + MP52 (20 ng/ml) 7 NPE + F (20 ng/ml) + E (20 ng/ml)Condition 1B + BMP7 (20 ng/ml) 8 NPE + F (20 ng/ml) + E (20 ng/ml)Condition 1B + GDNF (20 ng/ml) 9 NPE + F (20 ng/ml) + E (20 ng/ml)Condition 2B + MP52 (20 ng/ml) 10 NPE + F (20 ng/ml) + E (20 ng/ml)Condition 2B + BMP7 (20 ng/ml) 11 NPE + F (20 ng/ml) + E (20 ng/ml)Condition 2B + GDNF (20 ng/ml) 12 NPE + F (20 ng/ml) + E (20 ng/ml)Condition 3B + MP52 (20 ng/ml) 13 NPE + F (20 ng/ml) + E (20 ng/ml)Condition 3B + BMP7 (20 ng/ml) 14 NPE + F (20 ng/ml) + E (20 ng/ml)Condition 3B + GDNF (20 ng/ml) 15 NPE + F (20 ng/ml) + E (20 ng/ml)NPE + MP52 (20 ng/ml) 16 NPE + F (20 ng/ml) + E (20 ng/ml) NPE + BMP7(20 ng/ml) 17 NPE + F (20 ng/ml) + E (20 ng/ml) NPE + GDNF (20 ng/ml)

Multiple growth factor protocol. Umbilicus-derived cells (P11; (042203))were thawed and culture expanded in Growth Medium at 5,000 cells/cm²until sub-confluence (75%) was reached. Cells were then trypsinized andseeded at 2,000 cells/cm², onto 24 well laminin-coated plates (BDBiosciences) in the presence of NPE+F (20 nanograms/milliliter)+E(20nanograms/milliliter). In addition, some wells contained NPE+F+E+2% FBSor 10% FBS. After four days of “pre-differentiation” conditions, allmedia were removed and samples were switched to NPE medium supplementedwith sonic hedgehog (SHH; 200 nanograms/milliliter; Sigma, St. Louis,Mo.), FGF8 (100 nanograms/milliliter; Peprotech), BDNF (40nanograms/milliliter; Sigma), GDNF (20 nanograms/milliliter; Sigma), andretinoic acid (1 μM; Sigma). Seven days post medium change, cultureswere fixed with ice-cold 4% (w/v) paraformaldehyde (Sigma) for 10minutes at room temperature, and stained for human nestin, GFAP, TuJ1,desmin, and alpha-smooth muscle actin expression.

Neural progenitor co-culture protocol. Adult rat hippocampal progenitors(062603) were plated as neurospheres or single cells (10,000 cells/well)onto laminin-coated 24 well dishes (BD Biosciences) in NPE+F (20nanograms/milliliter)+E (20 nanograms/milliliter).

Separately, umbilicus-derived cells (042203) P11 and placenta-derivedcells (022803) P11 were thawed and culture expanded in NPE+F (20nanograms/milliliter)+E (20 nanograms/milliliter) at 5,000 cells/cm² fora period of 48 hours. Cells were then trypsinized and seeded at 2,500cells/well onto existing cultures of neural progenitors. At that time,existing medium was exchanged for fresh medium. Four days later,cultures were fixed with ice-cold 4% (w/v) paraformaldehyde (Sigma) for10 minutes at room temperature, and stained for human nuclear protein(hNuc; Chemicon) (Table NU1-1 above) to identify PPDCs.

Immunocytochemistry. Immunocytochemistry was performed using theantibodies listed in Table NU1-1. Cultures were washed withphosphate-buffered saline (PBS) and exposed to a protein blockingsolution containing PBS, 4% (v/v) goat serum (Chemicon, Temecula,Calif.), and 0.3% (v/v) Triton (Triton X-100; Sigma) for 30 minutes toaccess intracellular antigens. Primary antibodies, diluted in blockingsolution, were then applied to the cultures for a period of 1 hour atroom temperature. Next, primary antibodies solutions were removed andcultures washed with PBS prior to application of secondary antibodysolutions (1 hour at room temperature) containing blocking solutionalong with goat anti-mouse IgG—Texas Red (1:250; Molecular Probes,Eugene, Oreg.) and goat anti-rabbit IgG—Alexa 488 (1:250; MolecularProbes). Cultures were then washed and 10 micromolar DAPI (MolecularProbes) applied for 10 minutes to visualize cell nuclei.

Following immunostaining, fluorescence was visualized using theappropriate fluorescence filter on an Olympus inverted epi-fluorescentmicroscope (Olympus, Melville, N.Y.). In all cases, positive stainingrepresented fluorescence signal above control staining where the entireprocedure outlined above was followed with the exception of applicationof a primary antibody solution. Representative images were capturedusing a digital color videocamera and ImagePro software (MediaCybernetics, Carlsbad, Calif.). For triple-stained samples, each imagewas taken using only one emission filter at a time. Layered montageswere then prepared using Adobe Photoshop software (Adobe, San Jose,Calif.).

Results

Woodbury-Black protocol. (A) Upon incubation in this neural inductioncomposition, all cell types transformed into cells with bipolarmorphologies and extended processes. Other larger non-bipolarmorphologies were also observed. Furthermore, the induced cellpopulations stained positively for nestin, a marker of multipotentneural stem and progenitor cells.

(B) When repeated on tissue culture plastic (TCP) dishes, nestinexpression was not observed unless laminin was pre-adsorbed to theculture surface. To further assess whether nestin-expressing cells couldthen go on to generate mature neurons, PPDCs and fibroblasts wereexposed to NPE+RA (1 μM), a media composition known to induce thedifferentiation of neural stem and progenitor cells into such cells(2,3,4). Cells were stained for TuJ1, a marker for immature and matureneurons, GFAP, a marker of astrocytes, and nestin. Under no conditionswas TuJ1 detected, nor were cells with neuronal morphology observed,suggesting that neurons were not generated in the short term.Furthermore, nestin and GFAP were no longer expressed by PPDCs, asdetermined by immunocytochemistry.

Two-stage differentiation. Umbilicus and placenta PPDC isolates (as wellas human fibroblasts and rodent neural progenitors as negative andpositive control cell types, respectively) were plated on laminin(neural promoting)-coated dishes and exposed to 13 different growthconditions (and two control conditions) known to promote differentiationof neural progenitors into neurons and astrocytes. In addition, twoconditions were added to examine the influence of GDF5, and BMP7 on PPDCdifferentiation. Generally, a two-step differentiation approach wastaken, where the cells were first placed in neural progenitor expansionconditions for a period of 6 days, followed by full differentiationconditions for 7 days. Morphologically, both umbilicus- andplacenta-derived cells exhibited fundamental changes in cell morphologythroughout the time-course of this procedure. However, neuronal orastrocytic-shaped cells were not observed except for in control, neuralprogenitor-plated conditions. Immunocytochemistry, negative for humannestin, TuJ1, and GFAP confirmed the morphological observations.

Multiple growth factors. Following one week's exposure to a variety ofneural differentiation agents, cells were stained for markers indicativeof neural progenitors (human nestin), neurons (TuJ1), and astrocytes(GFAP). Cells grown in the first stage in non-serum containing media haddifferent morphologies than those cells in serum containing (2% or 10%)media, indicating potential neural differentiation. Specifically,following a two step procedure of exposing umbilicus-derived cells toEGF and bFGF, followed by SHH, FGF8, GDNF, BDNF, and retinoic acid,cells showed long extended processes similar to the morphology ofcultured astrocytes. When 2% FBS or 10% FBS was included in the firststage of differentiation, cell number was increased and cell morphologywas unchanged from control cultures at high density. Potential neuraldifferentiation was not evidenced by immunocytochemical analysis forhuman nestin, TuJ1, or GFAP.

Neural progenitor and PPDC co-culture. PPDCs were plated onto culturesof rat neural progenitors seeded two days earlier in neural expansionconditions (NPE+F+E). While visual confirmation of plated PPDCs provedthat these cells were plated as single cells, human-specific nuclearstaining (hNuc) 4 days post-plating (6 days total) showed that theytended to ball up and avoid contact with the neural progenitors.Furthermore, where PPDCs attached, these cells spread out and appearedto be innervated by differentiated neurons that were of rat origin,suggesting that the PPDCs may have differentiated into muscle cells.This observation was based upon morphology under phase contrastmicroscopy. Another observation was that typically large cell bodies(larger than neural progenitors) possessed morphologies resemblingneural progenitors, with thin processes spanning out in multipledirections. HNuc staining (found in one half of the cell's nucleus)suggested that in some cases these human cells may have fused with ratprogenitors and assumed their phenotype. Control wells containing onlyneural progenitors had fewer total progenitors and apparentdifferentiated cells than did co-culture wells containing umbilicus orplacenta PPDCs, further indicating that both umbilicus- andplacenta-derived cells influenced the differentiation and behavior ofneural progenitors, either by release of chemokines and cytokines, or bycontact-mediated effects.

Summary. Multiple protocols were conducted to determine the short termpotential of PPDCs to differentiate into neural lineage cells. Theseincluded phase contrast imaging of morphology in combination withimmunocytochemistry for nestin, TuJ1, and GFAP, proteins associated withmultipotent neural stem and progenitor cells, immature and matureneurons, and astrocytes, respectively. Evidence was observed to suggestthat neural differentiation occurred in certain instances in theseshort-term protocols.

Several notable observations were made in co-cultures of PPDCs withneural progenitors. This approach, using human PPDCs along with axenogeneic cell type allowed for absolute determination of the origin ofeach cell in these cultures. First, some cells were observed in thesecultures where the cell cytoplasm was enlarged, with neurite-likeprocesses extending away from the cell body, yet only half of the bodylabeled with hNuc protein. Those cells may have been human PPDCs thathad differentiated into neural lineage cells or they may have been PPDCsthat had fused with neural progenitors. Second, it appeared that neuralprogenitors extended neurites to PPDCs in a way that indicates theprogenitors differentiated into neurons and innervated the PPDCs. Third,cultures of neural progenitors and PPDCs had more cells of rat originand larger amounts of differentiation than control cultures of neuralprogenitors alone, further indicating that plated PPDCs provided solublefactors and or contact-dependent mechanisms that stimulated neuralprogenitor survival, proliferation, and/or differentiation.

References for Example 13

(1) Woodbury, D. et al. (2000). J. Neurosci. Research. 61(4): 364-70.

(2) Jang, Y. K. et al. (2004). J. Neurosci. Research. 75(4): 573-84.

(3) Jones-Villeneuve, E. M. et al. (1983). Mol Cel Biol. 3(12): 2271-9.

(4) Mayer-Proschel, M. et al. (1997). Neuron. 19(4): 773-85.

EXAMPLE 14 Long-Term Neural Differentiation of Postpartum-Derived Cells

The ability of umbilicus and placenta-derived cells (collectivelypostpartum-derived cells or PPDCs) to undergo long-term differentiationinto neural lineage cells was evaluated.

Materials & Methods

Isolation and Expansion of PPDCs. PPDCs were isolated and expanded asdescribed in previous Examples.

PPDC Cell Thaw and Plating. Frozen aliquots of PPDCs (umbilicus (022803)P11; (042203) P11; (071003) P12; placenta (101503) P7) previously grownin Growth Medium were thawed and plated at 5,000 cells/cm² in T-75flasks coated with laminin (BD, Franklin Lakes, N.J.) in Neurobasal-Amedium (Invitrogen, Carlsbad, Calif.) containing B27 (B27 supplement,Invitrogen), L-glutamine (4 mM), and Penicillin/Streptomycin (10milliliters), the combination of which is herein referred to as NeuralProgenitor Expansion (NPE) media. NPE media was further supplementedwith bFGF (20 nanograms/milliliter, Peprotech, Rocky Hill, N.J.) and EGF(20 nanograms/milliliter, Peprotech, Rocky Hill, N.J.), herein referredto as NPE+bFGF+EGF.

Control Cell Plating. In addition, adult human dermal fibroblasts (P11,Cambrex, Walkersville, Md.) and mesenchymal stem cells (P5, Cambrex)were thawed and plated at the same cell seeding density onlaminin-coated T-75 flasks in NPE+bFGF+EGF. As a further control,fibroblasts, umbilicus, and placenta PPDCs were grown in Growth Mediumfor the period specified for all cultures.

Cell Expansion. Media from all cultures were replaced with fresh mediaonce a week and cells observed for expansion. In general, each culturewas passaged one time over a period of one month because of limitedgrowth in NPE+bFGF+EGF.

Immunocytochemistry. After a period of one month, all flasks were fixedwith cold 4% (w/v) paraformaldehyde (Sigma) for 10 minutes at roomtemperature. Immunocytochemistry was performed using antibodies directedagainst TuJ1 (BIII Tubulin; 1:500; Sigma, St. Louis, Mo.) and GFAP(glial fibrillary acidic protein; 1:2000; DakoCytomation, Carpinteria,Calif.). Briefly, cultures were washed with phosphate-buffered saline(PBS) and exposed to a protein blocking solution containing PBS, 4%(v/v) goat serum (Chemicon, Temecula, Calif.), and 0.3% (v/v) Triton(Triton X-100; Sigma) for 30 minutes to access intracellular antigens.Primary antibodies, diluted in blocking solution, were then applied tothe cultures for a period of 1 hour at room temperature. Next, primaryantibodies solutions were removed and cultures washed with PBS prior toapplication of secondary antibody solutions (1 hour at room temperature)containing block along with goat anti-mouse IgG—Texas Red (1:250;Molecular Probes, Eugene, Oreg.) and goat anti-rabbit IgG—Alexa 488(1:250; Molecular Probes). Cultures were then washed and 10 micromolarDAPI (Molecular Probes) applied for 10 minutes to visualize cell nuclei.

Following immunostaining, fluorescence was visualized using theappropriate fluorescence filter on an Olympus inverted epi-fluorescentmicroscope (Olympus, Melville, N.Y.). In all cases, positive stainingrepresented fluorescence signal above control staining where the entireprocedure outlined above was followed with the exception of applicationof a primary antibody solution. Representative images were capturedusing a digital color videocamera and ImagePro software (MediaCybernetics, Carlsbad, Calif.). For triple-stained samples, each imagewas taken using only one emission filter at a time. Layered montageswere then prepared using Adobe Photoshop software (Adobe, San Jose,Calif.).

TABLE 14-1 Summary of Primary Antibodies Used Antibody ConcentrationVendor TuJ1 (BIII Tubulin) 1:500 Sigma, St. Louis, MO GFAP 1:2000DakoCytomation, Carpinteria, CA

Results

NPE+bFGF+EGF media slows proliferation of PPDCs and alters theirmorphology. Immediately following plating, a subset of PPDCs attached tothe culture flasks coated with laminin. This may have been due to celldeath as a function of the freeze/thaw process or because of the newgrowth conditions. Cells that did attach adopted morphologies differentfrom those observed in Growth Media.

Upon confluence, cultures were passaged and observed for growth. Verylittle expansion took place of those cells that survived passage. Atthis point, very small cells with no spread morphology and withphase-bright characteristics began to appear in cultures ofumbilicus-derived cells. These areas of the flask were followed overtime. From these small cells, bifurcating processes emerged withvaricosities along their lengths, features very similar to previouslydescribed PSA-NCAM+neuronal progenitors and TuJ1+immature neuronsderived from brain and spinal cord (1, 2). With time, these cells becamemore numerous, yet still were only found in clones.

Clones of umbilicus-derived cells express neuronal proteins. Cultureswere fixed at one month post-thawing/plating and stained for theneuronal protein TuJ1 and GFAP, an intermediate filament found inastrocytes. While all control cultures grown in Growth Medium and humanfibroblasts and MSCs grown in NPE+bFGF+EGF medium were found to beTuJ1-/GFAP-, TuJ1 was detected in the umbilicus and placenta PPDCs.Expression was observed in cells with and without neuronal-likemorphologies. No expression of GFAP was observed in either culture. Thepercentage of cells expressing TuJ1 with neuronal-like morphologies wasless than or equal to 1% of the total population (n=3 umbilicus-derivedcell isolates tested). While not quantified, the percentage ofTujl+cells without neuronal morphologies was higher in umbilicus-derivedcell cultures than placenta-derived cell cultures. These resultsappeared specific as age-matched controls in Growth Medium did notexpress TuJ1.

Summary. Methods for generating differentiated neurons (based on TuJ1expression and neuronal morphology) from umbilicus-derived cells weredeveloped. While expression for TuJ1 was not examined earlier than onemonth in vitro, it is clear that at least a small population ofumbilicus-derived cells can give rise to neurons either through defaultdifferentiation or through long-term induction following one month'sexposure to a minimal media supplemented with L-glutamine, basic FGF,and EGF.

References for Example 14

(1) Mayer-Proschel, M. et al. (1997). Neuron. 19(4): 773-85.

(2) Yang, H. et al. (2000). PNAS. 97(24): 13366-71.

EXAMPLE 15 PPDC Trophic Factors for Neural Progenitor Support

The influence of umbilicus- and placenta-derived cells (collectivelypostpartum-derived cells or PPDCs) on adult neural stem and progenitorcell survival and differentiation through non-contact dependent(trophic) mechanisms was examined.

Materials & Methods

Adult neural stem and progenitor cell isolation. Fisher 344 adult ratswere sacrificed by CO₂ asphyxiation followed by cervical dislocation.Whole brains were removed intact using bone rongeurs and hippocampustissue dissected based on coronal incisions posterior to the motor andsomatosensory regions of the brain (Paxinos, G. & Watson, C. 1997. TheRat Brain in Stereotaxic Coordinates). Tissue was washed in Neurobasal-Amedium (Invitrogen, Carlsbad, Calif.) containing B27 (B27 supplement;Invitrogen), L-glutamine (4 mM; Invitrogen), and penicillin/streptomycin(Invitrogen), the combination of which is herein referred to as NeuralProgenitor Expansion (NPE) medium. NPE medium was further supplementedwith bFGF (20 nanograms/milliliter, Peprotech, Rocky Hill, N.J.) and EGF(20 nanograms/milliliter, Peprotech, Rocky Hill, N.J.), herein referredto as NPE+bFGF+EGF.

Following wash, the overlying meninges were removed, and the tissueminced with a scalpel. Minced tissue was collected and trypsin/EDTA(Invitrogen) added as 75% of the total volume. DNAse (100 microlitersper 8 milliliters total volume, Sigma, St. Louis, Mo.) was also added.Next, the tissue/media was sequentially passed through an 18 gaugeneedle, 20 gauge needle, and finally a 25 gauge needle one time each(all needles from Becton Dickinson, Franklin Lakes, N.J.). The mixturewas centrifuged for 3 minutes at 250 g. Supernatant was removed, freshNPE+bFGF+EGF was added and the pellet resuspended. The resultant cellsuspension was passed through a 40 micrometer cell strainer (BectonDickinson), plated on laminin-coated T-75 flasks (Becton Dickinson) orlow cluster 24-well plates (Becton Dickinson), and grown in NPE+bFGF+EGFmedia until sufficient cell numbers were obtained for the studiesoutlined.

PPDC plating. Postpartum-derived cells (umbilicus (022803) P12, (042103)P12, (071003) P12; placenta (042203) P12) previously grown in GrowthMedium were plated at 5,000 cells/transwell insert (sized for 24 wellplate) and grown for a period of one week in Growth Medium in inserts toachieve confluence.

Adult neural progenitor plating. Neural progenitors, grown asneurospheres or as single cells, were seeded onto laminin-coated 24 wellplates at an approximate density of 2,000 cells/well in NPE+bFGF+EGF fora period of one day to promote cellular attachment. One day later,transwell inserts containing postpartum cells were added according tothe following scheme:

-   -   (1) Transwell (umbilicus-derived cells in Growth Media, 200        microliters)+neural progenitors (NPE+bFGF+EGF, 1 milliliter)    -   (2) Transwell (placenta-derived cells in Growth Media, 200        microliters)+neural progenitors (NPE+bFGF+EGF, 1 milliliter)    -   (3) Transwell (adult human dermal fibroblasts [IF1853; Cambrex,        Walkersville, Md.] P12 in Growth Media, 200 microliters)+neural        progenitors (NPE+bFGF+EGF, 1 milliliter)    -   (4) Control: neural progenitors alone (NPE+bFGF+EGF, 1        milliliter)    -   (5) Control: neural progenitors alone (NPE only, 1 milliliter)

Immunocytochemistry. After 7 days in co-culture, all conditions werefixed with cold 4% (w/v) paraformaldehyde (Sigma) for a period of 10minutes at room temperature. Immunocytochemistry was performed usingantibodies directed against the epitopes listed in Table 15-1. Briefly,cultures were washed with phosphate-buffered saline (PBS) and exposed toa protein blocking solution containing PBS, 4% (v/v) goat serum(Chemicon, Temecula, Calif.), and 0.3% (v/v) Triton (Triton X-100;Sigma) for 30 minutes to access intracellular antigens. Primaryantibodies, diluted in blocking solution, were then applied to thecultures for a period of 1 hour at room temperature. Next, primaryantibodies solutions were removed and cultures washed with PBS prior toapplication of secondary antibody solutions (1 hour at room temperature)containing blocking solution along with goat anti-mouse IgG—Texas Red(1:250; Molecular Probes, Eugene, Oreg.) and goat anti-rabbit IgG—Alexa488 (1:250; Molecular Probes). Cultures were then washed and 10micromolar DAPI (Molecular Probes) applied for 10 minutes to visualizecell nuclei.

Following immunostaining, fluorescence was visualized using theappropriate fluorescence filter on an Olympus inverted epi-fluorescentmicroscope (Olympus, Melville, N.Y.). In all cases, positive stainingrepresented fluorescence signal above control staining where the entireprocedure outlined above was followed with the exception of applicationof a primary antibody solution. Representative images were capturedusing a digital color videocamera and ImagePro software (MediaCybernetics, Carlsbad, Calif.). For triple-stained samples, each imagewas taken using only one emission filter at a time. Layered montageswere then prepared using Adobe Photoshop software (Adobe, San Jose,Calif.).

TABLE 15-1 Summary of Primary Antibodies Used Antibody ConcentrationVendor Rat 401 (nestin) 1:200 Chemicon, Temecula, CA TuJ1 (BIII Tubulin)1:500 Sigma, St. Louis, MO Tyrosine hydroxylase 1:1000 Chemicon (TH)GABA 1:400 Chemicon GFAP 1:2000 DakoCytomation, Carpinteria, CA MyelinBasic Protein 1:400 Chemicon (MBP)

Quantitative analysis of neural progenitor differentiation.Quantification of hippocampal neural progenitor differentiation wasexamined. A minimum of 1000 cells were counted per condition or if less,the total number of cells observed in that condition. The percentage ofcells positive for a given stain was assessed by dividing the number ofpositive cells by the total number of cells as determined by DAPI(nuclear) staining.

Mass spectrometry analysis & 2D gel electrophoresis. In order toidentify unique, secreted factors as a result of co-culture, conditionedmedia samples taken prior to culture fixation were frozen down at −80°C. overnight. Samples were then applied to ultrafiltration spin devices(MW cutoff 30 kD). Retentate was applied to immunoaffinitychromatography (anti-Hu-albumin; IgY) (immunoaffinity did not removealbumin from the samples). Filtrate was analyzed by MALDI. The passthrough was applied to Cibachron Blue affinity chromatography. Sampleswere analyzed by SDS-PAGE and 2D gel electrophoresis.

Results

PPDC co-culture stimulates adult neural progenitor differentiation.Following culture with umbilicus- or placenta-derived cells, co-culturedneural progenitor cells derived from adult rat hippocampus exhibitedsignificant differentiation along all three major lineages in thecentral nervous system. This effect was clearly observed after five daysin co-culture, with numerous cells elaborating complex processes andlosing their phase bright features characteristic of dividing progenitorcells. Conversely, neural progenitors grown alone in the absence of bFGFand EGF appeared unhealthy and survival was limited.

After completion of the procedure, cultures were stained for markersindicative of undifferentiated stem and progenitor cells (nestin),immature and mature neurons (TuJ1), astrocytes (GFAP), and matureoligodendrocytes (MBP). Differentiation along all three lineages wasconfirmed while control conditions did not exhibit significantdifferentiation as evidenced by retention of nestin-positive stainingamongst the majority of cells. While both umbilicus- andplacenta-derived cells induced cell differentiation, the degree ofdifferentiation for all three lineages was less in co-cultures withplacenta-derived cells than in co-cultures with umbilicus-derived cells.

The percentage of differentiated neural progenitors following co-culturewith umbilicus-derived cells was quantified (Table 15-2).Umbilicus-derived cells significantly enhanced the number of matureoligodendrocytes (MBP) (24.0% vs 0% in both control conditions).Furthermore, co-culture enhanced the number of GFAP+astrocytes and TuJ1+neurons in culture (47.2% and 8.7% respectively). These results wereconfirmed by nestin staining indicating that progenitor status was lostfollowing co-culture (13.4% vs 71.4% in control condition 4).

Though differentiation also appeared to be influenced by adult humanfibroblasts, such cells were not able to promote the differentiation ofmature oligodendrocytes nor were they able to generate an appreciablequantity of neurons. Though not quantified, fibroblasts did, however,appear to enhance the survival of neural progenitors.

TABLE 15-2 Quantification of progenitor differentiation in control vstranswell co-culture with umbilical-derived cells (E = EGF, F = bFGF)F + E/Umb F + E/F + E F + E/removed Antibody [Cond. 1] [Cond. 4] [Cond.5] TuJ1 8.7%  2.3%  3.6% GFAP 47.2% 30.2% 10.9% MBP 23.0%   0%   0%Nestin 13.4% 71.4% 39.4%

Identification of unique compounds. Conditioned media from umbilicus-and placenta-derived co-cultures, along with the appropriate controls(NPE media±1.7% serum, media from co-culture with fibroblasts), wereexamined for differences. Potentially unique compounds were identifiedand excised from their respective 2D gels.

Summary. Co-culture of adult neural progenitor cells with umbilicus orplacenta PPDCs results in differentiation of those cells. Resultspresented in this example indicate that the differentiation of adultneural progenitor cells following co-culture with umbilicus-derivedcells is particularly profound. Specifically, a significant percentageof mature oligodendrocytes was generated in co-cultures ofumbilicus-derived cells. In view of the lack of contact between theumbilicus-derived cells and the neural progenitors, this result appearsto be a function of soluble factors released from the umbilicus-derivedcells (trophic effect).

Several other observations were made. First, there were very few cellsin the control condition where EGF and bFGF were removed. Most cellsdied and on average, there were about 100 cells or fewer per well.Second, it is to be expected that there would be very littledifferentiation in the control condition where EGF and bFGF was retainedin the medium throughout, since this is normally an expansion medium.While approximately 70% of the cells were observed to retain theirprogenitor status (nestin+), about 30% were GFAP+(indicative ofastrocytes). This may be due to the fact that such significant expansionoccurred throughout the course of the procedure that contact betweenprogenitors induced this differentiation (Song, H. et al. 2002. Nature417:6884 39-44).

EXAMPLE 16 Transplantation of Postpartum-Derived Cells

Cells derived from the postpartum umbilicus and placenta are useful forregenerative therapies. The tissue produced by postpartum-derived cells(PPDCs) transplanted into SCID mice with a biodegradable material wasevaluated. The materials evaluated were Vicryl non-woven, 35/65 PCL/PGAfoam, and RAD 16 self-assembling peptide hydrogel.

Methods & Materials

Cell Culture. Placenta- and umbilicus-derived cells were grown in GrowthMedium (DMEM-low glucose (Gibco, Carlsbad Calif.), 15% (v/v) fetalbovine serum (Cat. #SH30070.03; Hyclone, Logan, Utah), 0.001% (v/v)betamercaptoethanol (Sigma, St. Louis, Mo.), penicillin/streptomycin(Gibco)) in a gelatin-coated flasks.

Sample Preparation. One million viable cells were seeded in 15microliters Growth Medium onto 5 mm diameter, 2.25 mm thick Vicrylnon-woven scaffolds (64.33 milligrams/cc; Lot#3547-47-1) or 5 mmdiameter 35/65 PCL/PGA foam (Lot# 3415-53). Cells were allowed to attachfor two hours before adding more Growth Medium to cover the scaffolds.Cells were grown on scaffolds overnight. Scaffolds without cells werealso incubated in medium.

RAD16 self-assembling peptides (3D Matrix, Cambridge, Mass. under amaterial transfer agreement) was obtained as a sterile 1% (w/v) solutionin water, which was mixed 1:1 with 1×10⁶ cells in 10% (w/v) sucrose(Sigma, St Louis, Mo.), 10 mM HEPES in Dulbecco's modified medium (DMEM;Gibco) immediately before use. The final concentration of cells in RAD16hydrogel was 1×10⁶ cells/100 microliters.

TEST MATERIAL (N=4/Rx)

-   -   1. Vicryl non-woven+1×10⁶ umbilicus-derived cells    -   2. 35/65 PCL/PGA foam+1×10⁶ umbilicus-derived cells    -   3. RAD 16 self-assembling peptide+1×10⁶ umbilicus-derived cells    -   4. Vicryl non-woven+1×10⁶ placenta-derived cells    -   5. 35/65 PCL/PGA foam+1×10⁶ placenta-derived cells    -   6. RAD 16 self-assembling peptide+1×10⁶ placenta-derived cells    -   7. 35/65 PCL/PGA foam    -   8. Vicryl non-woven

Animal Preparation. The animals were handled and maintained inaccordance with the current requirements of the Animal Welfare Act.Compliance with the above Public Laws were accomplished by adhering tothe Animal Welfare regulations (9 CFR) and conforming to the currentstandards promulgated in the Guide for the Care and Use of LaboratoryAnimals, 7th edition.

Mice (Mus Musculus)/Fox Chase SCID/Male (Harlan Sprague Dawley, Inc.,Indianapolis, Ind.), 5 weeks of age. All handling of the SCID mice tookplace under a hood. The mice were individually weighed and anesthetizedwith an intraperitoneal injection of a mixture of 60 milligrarns/kgKETASET (ketamine hydrochloride, Aveco Co., Inc., Fort Dodge, Iowa) and10 milligrams/kg ROMPUN (xylazine, Mobay Corp., Shawnee, Kans.) andsaline. After induction of anesthesia, the entire back of the animalfrom the dorsal cervical area to the dorsal lumbosacral area was clippedfree of hair using electric animal clippers. The area was then scrubbedwith chlorhexidine diacetate, rinsed with alcohol, dried, and paintedwith an aqueous iodophor solution of 1% available iodine. Ophthalmicointment was applied to the eyes to prevent drying of the tissue duringthe anesthetic period.

Subcutaneous Implantation Technique. Four skin incisions, eachapproximately 1.0 cm in length, were made on the dorsum of the mice. Twocranial sites were located transversely over the dorsal lateral thoracicregion, about 5-mm caudal to the palpated inferior edge of the scapula,with one to the left and one to the right of the vertebral column.Another two were placed transversely over the gluteal muscle area at thecaudal sacro-lumbar level, about 5-mm caudal to the palpated iliaccrest, with one on either side of the midline. Implants were randomlyplaced in these sites in accordance with the experimental design. Theskin was separated from the underlying connective tissue to make a smallpocket and the implant placed (or injected for RAD16) about 1-cm caudalto the incision. The appropriate test material was implanted into thesubcutaneous space. The skin incision was closed with metal clips.

Animal Housing. Mice were individually housed in microisolator cagesthroughout the course of the study within a temperature range of 64°F.-79° F. and relative humidity of 30% to 70%, and maintained on anapproximate 12 hour light/12 hour dark cycle. The temperature andrelative humidity were maintained within the stated ranges to thegreatest extent possible. Diet consisted of Irradiated Pico Mouse Chow5058 (Purina Co.) and water fed ad libitum.

Mice were euthanized at their designated intervals by carbon dioxideinhalation. The subcutaneous implantation sites with their overlyingskin were excised and frozen for histology.

Histology. Excised skin with implant was fixed with 10% neutral bufferedformalin (Richard-Allan Kalamazoo, Mich.). Samples with overlying andadjacent tissue were centrally bisected, paraffin-processed, andembedded on cut surface using routine methods. Five-micron tissuesections were obtained by microtome and stained with hematoxylin andeosin (Poly Scientific Bay Shore, N.Y.) using routine methods.

Results

There was minimal ingrowth of tissue into foams (without cells)implanted subcutaneously in SCID mice after 30 days. In contrast therewas extensive tissue fill in foams implanted with umbilical-derivedcells or placenta-derived cells. Some tissue ingrowth was observed inVicryl non-woven scaffolds. Non-woven scaffolds seeded with umbilicus-or placenta-derived cells showed increased matrix deposition and matureblood vessels.

Summary. Synthetic absorbable non-woven/foam discs (5.0 mm diameter×1.0mm thick) or self-assembling peptide hydrogel were seeded with eithercells derived from human umbilicus or placenta and implantedsubcutaneously bilaterally in the dorsal spine region of SCID mice. Theresults demonstrated that postpartum-derived cells could dramaticallyincrease good quality tissue formation in biodegradable scaffolds.

EXAMPLE 17 Use of Postpartum-Derived Cells in Nerve Repair

Retinal ganglion cell (RGC) lesions have been extensively used as modelsfor various repair strategies in the adult mammalian CNS. It has beendemonstrated that retrobulbar section of adult rodent RGC axons resultsin abortive sprouting (Zeng et al., 1995) and progressive death of theparent cell population (Villegas-Perez et al., 1993). Numerous studieshave demonstrated the stimulatory effects of various exogenous andendogenous factors on the survival of axotomized RGC's and regenerationof their axons (Yip and So, 2000; Fischer et al., 2001). Furthermore,other studies have demonstrated that cell transplants can be used topromote regeneration of severed nerve axons (Li et al., 2003;Ramon-Cueto et al., 2000). Thus, these and other studies havedemonstrated that cell based therapy can be utilized for the treatmentof neural disorders that affect the spinal cord, peripheral nerves,pudendal nerves, optic nerves or other diseases/trauma due to injury inwhich nervous damage can occur.

Self-assembling peptides (PuraMatrix™, U.S. Pat. Nos. 5,670,483,5,955,343, US/PCT applications US2002/0160471, WO02/062969) have beendeveloped to act as a scaffold for cell-attachment to encapsulate cellsin 3-D, plate cells in 2-D coatings, or as microcarriers in suspensioncultures. Three-dimensional cell culture has required eitheranimal-derived materials (mouse sarcoma extract), with their inherentreproducibility and cell signaling issues, or much larger syntheticscaffolds, which fail to approximate the physical nanometer-scale andchemical attributes of native ECM. RAD 16 (NH2—(RADA)₃—COOH) and KLD(NH2-(KLDL)₃—COOH) are synthesized in small (RAD16 is 5 nanometers)oligopeptide fragments that self-assemble into nanofibers on a scalesimilar to the in vivo extracellular matrix (ECM) (3D Matrix, IncCambridge, Mass.). The self-assembly is initiated by mono- or di-valentcations found in culture media or the physiological environment. In theprotocols described in this example, RAD 16 was used as a microcarrierfor the implantation of postpartum cells into the ocular defect. In thisexample, it is demonstrated that transplants of postpartum-derived cellsPPDCs) can provide efficacy in an adult rat optic nerve axonalregeneration model.

Methods & Materials

Cells. Cultures of human adult PPDCs (umbilicus and placenta) andfibroblast cells (passage 10) were expanded for 1 passage. All cellswere initially seeded at 5,000 cells/cm² on gelatin-coated T75 flasks inGrowth Medium with 100 Units per milliliter penicillin, 100 microgramsper milliliter streptomycin, 0.25 micrograms per milliliter amphotericinB (Invitrogen, Carlsbad, Calif.). At passage 11 cells were trypsinizedand viability was determined using trypan blue staining. Briefly, 50microliters of cell suspension was combined with 50 microliters of 0.04%w/v trypan blue (Sigma, St. Louis Mo.) and the viable cell number, wasestimated using a hemocytometer. Cells were then washed three times insupplement free-Leibovitz's L-15 medium (Invitrogen, Carlsbad, Calif.).Cells were then suspended at a concentration of 200,000 cells in 25microliters of RAD-16 (3DM Inc., Cambridge, Mass.) which was bufferedand made isotonic as per manufacturer's recommendations. One hundredmicroliters of supplement free Leibovitz's L-15 medium was added abovethe cell/matrix suspension to keep it wet till use. These cell/matrixcultures were maintained under standard atmospheric conditions untiltransplantation occurred. At the point of transplantation the excessmedium was removed.

Animals and Surgery. Long Evans female rats (220-240 gram body weight)were used. Under intraperitoneal tribromoethanol anesthesia (20milligram/100 grams body weight), the optic nerve was exposed, and theoptic sheath was incised intraorbitally at approximately 2 millimetersfrom the optic disc, the nerve was lifted from the sheath to allowcomplete transsection with fine scissors (Li et al., 2003). Thecompleteness of transsection was confirmed by visually observingcomplete separation of the proximal and distal stumps. The control groupconsisted of lesioned rats without transplants. In transplant ratscultured postpartum cells seeded in RAD-16 were inserted between theproximal and distal stumps using a pair of microforceps. Approximately75,000 cells in RAD-16 were implanted into the severed optic nerve.Cell/matrix was smeared into the severed cut using a pair of finemicroforceps. The severed optic nerve sheath was closed with 10/0 blackmonofilament nylon (Ethicon, Inc., Edinburgh, UK). Thus, the gap wasclosed by drawing the cut proximal and distal ends of the nerve inproximity with each other.

After cell injections were performed, animals were injected withdexamethasone (2 milligrams/kilogram) for 10 days post transplantation.For the duration of the study, animals were maintained on oralcyclosporine A (210 milligrams/liter of drinking water; resulting bloodconcentration: 250-300 micrograms/liter) (Bedford Labs, Bedford, Ohio)from 2 days pre-transplantation until end of the study. Food and waterwere available ad libitum. Animals were sacrificed at either 30 or 60days posttransplantation.

CTB Application. Three days before animals were sacrificed, underanesthesia, a glass micropipette with a 30-50 millimeter tip wasinserted tangentially through the sclera behind the lens, and two 4-5microliter aliquots of a 1% retrograde tracer-cholera toxin B (CTB)aqueous solution (List Biologic, Campbell, Calif.) was injected into thevitreous. Animals were perfused with fixative and optic nerves werecollected in the same fixative for 1 hour. The optic nerves weretransferred into sucrose overnight. Twenty micrometer cryostat sectionswere incubated in 0.1 molar glycine for 30 minutes and blocked in a PBSsolution containing 2.5% bovine serum albumin (BSA) (Boeringer Mannheim,Mannheim, Germany) and 0.5% triton X-100 (Sigma, St. Louis, Mo.),followed by a solution containing goat anti-CTB antibody (List Biologic,Campbell, Calif.) diluted 1:4000 in a PBS containing 2% normal rabbitserum (NRS) (Invitrogen, Carlsbad, Calif.), 2.5% BSA, and 2% TritonX-100 (Sigma, St. Louis, Mo.) in PBS, and incubated in biotinylatedrabbit anti-goat IgG antibody (Vector Laboratories, Burlinghame, Calif.)diluted 1:200 in 2% Triton-X100 in PBS for 2 hours at room temperature.This was followed by staining in 1:200 streptavidin-green (Alexa Flour438;Molecular Probes, Eugene, Oreg.) in PBS for 2 hours at roomtemperature. Stained sections were then washed in PBS and counterstainedwith propidium iodide for confocal microscopy.

Histology Preparation. Briefly, 5 days after CTB injection, rats wereperfused with 4% paraformaldehyde. Rats were given 4 cubic centimetersof urethane and were then perfused with PBS (0.1 molar) then with 4%Para formaldehyde. The spinal cord was cut and the bone removed from thehead to expose the colliculus. The colliculus was then removed andplaced in 4% paraformaldehyde. The eye was removed by cutting around theoutside of the eye and going as far back as possible. Care was given notto cut the optic nerve that lies on the underside of the eye. The eyewas removed and the muscles were cut exposing the optic nerve this wasthen placed in 4% paraformaldehyde.

Results

Lesions alone. One month after retrotubular section of the optic nerve,a number of CTB-labeled axons were identified in the nerve segmentattached to the retina. In the 200 micrometers nearest the cut, axonswere seen to emit a number of collaterals at right angles to the mainaxis and terminate as a neuromatous tangle at the cut surface. In thiscut between the proximal and distal stumps, the gap was observed to beprogressively bridged by a 2-3 millimeter segment of vascularizedconnective tissue; however, no axons were seen to advance into thisbridged area. Thus, in animals that received lesion alone no axonalgrowth was observed to reach the distal stump.

RAD-16 transplantation. Following transplantation of RAD-16 into thecut, visible ingrowth of vascularized connective tissue was observed.However, no axonal in growth was observed between the proximal anddistal stumps. The results demonstrate that application of RAD-16 aloneis not sufficient for inducing axonal regeneration in this situation.

Transplantation of postpartum-derived cells. Transplantation ofpostpartum-derived cells into the severed optic nerve stimulated opticnerve regrowth. Some regrowth was also observed in conditions in whichfibroblast cells were implanted, although this was minimal as comparedwith the regrowth observed with the transplanted placenta-derived cells.Optic nerve regrowth was observed in 4/5 animals transplanted withplacenta-derived cells, 3/6 animals transplanted with adult dermalfibroblasts and in 1/4 animals transplanted with umbilicus-derivedcells. In situations where regrowth was observed, CTB labeling confirmedregeneration of retinal ganglion cell axons, which were demonstrated topenetrate through the transplant area. GFAP labeling was also performedto determine the level of glial scarring. The GFAP expression wasintensified at the proximal stump with some immunostaining beingobserved through the reinervated graft.

Summary. These results demonstrate that transplanted human adultpostpartum-derived cells are able to stimulate and guide regeneration ofcut retinal ganglion cell axons.

References for Example 17

1) Zeng B Y, Anderson P N, Campbell G, Lieberman A R. 1995. J.Anat.186:495-508.

2) Villegas-Perez M P, Vidal-Sanz M, Bray G M, Aguayo A J. 1988. J.Neurosci.8:265-80.

3) Yip H K, So K F. 2000. Prog Retin Eye Res. 19: 559-75.

4) Fischer D, Heiduschka P, Thanos S. 2001. Exp Neurol. 172: 257-72.

5) Ramon-Cueto A, Cordero M I, Santos-Benito F F, Avila J. 2000. Neuron25: 425-35.

The present invention is not limited to the embodiments described andexemplified above. It is capable of variation and modification withinthe scope of the appended claims.

1. A method of treating a patient having nerve pathology caused bytraumatic nerve injury, said method comprising administering to thepatient one or more cells, wherein said one or more cells are isolatedfrom human postpartum umbilical cord substantially free of blood or areexpanded in culture from a cell isolated from human postpartum umbilicalcord substantially free of blood, in an amount effective to treat thenerve pathology, wherein said one or more cells self-renew and expand inculture and have the potential to differentiate into cells of at least aneural phenotype; require L-valine for growth; can grow in at leastabout 5% oxygen; do not produce CD117; and exhibit increased expression,relative to a human fibroblast or mesenchymal stem cell, of anendogenous gene encoding oxidized low density lipoprotein receptor 1,interleukin 8, or reticulon
 1. 2. The method of claim 1, wherein thecells are induced in vitro to differentiate into neural lineage cellsprior to administration.
 3. The method of claim 1, wherein the cells areadministered with at least one other cell type.
 4. The method of claim3, wherein the other cell type is an astrocyte, oligodendrocyte, neuron,neural progenitor, neural stem cell or other multipotent or pluripotentstem cell.
 5. The method of claim 3, wherein the at least one other celltype is administered simultaneously with, or before, or after, said oneor more cells.
 6. The method of claim 1, wherein the cells areadministered with at least one other agent, wherein said agent is a drugfor neural therapy, an anti-inflammatory agent, an anti-apoptotic agent,an antioxidant or a growth factor.
 7. The method of claim 6, wherein theat least one other agent is administered simultaneously with, or before,or after, said one or more cells.
 8. The method of claim 1, wherein thecells are administered at a predetermined site in the central orperipheral nervous system of the patient.
 9. The method of claim 1,wherein the cells are administered by injection or infusion.
 10. Themethod of claim 1, wherein the cells are administered encapsulatedwithin an implantable device.
 11. The method of claim 1, wherein thecells are administered by implantation of a matrix or scaffoldcontaining the cells.
 12. The method of claim 1, wherein the nervepathology is to a central nerve.
 13. The method of claim 1, wherein thenerve pathology is to a peripheral nerve.
 14. The method of claim 12,wherein the central nerve is the optic nerve.
 15. The method of claim 1wherein said one or more cells comprise the following characteristics:potential for at least about 40 doublings in culture; attachment andexpansion on a coated or uncoated tissue culture vessel, wherein saidcoated tissue culture vessel comprises a coating of gelatin, laminin,collagen, polyornithine, vitronectin, or fibronectin; production ofvimentin and alpha-smooth muscle actin; and production of CD10, CD13,CD44, CD73, and CD90.
 16. The method of claim 1 wherein the one or morecells express each of CD10, CD13, CD44, CD73, CD90, PDGFr-alpha, andHLA-A,B,C.
 17. The method of claim 1 wherein the one or more cells donot express any of CD31, CD34, CD45, CD141, or HLA-DR,DP,DQ.